Treating pain using selective antagonists of persistent sodium current

ABSTRACT

The present invention provides methods of treating chronic pain in a mammal by administering to the mammal an effective amount of a selective persistent sodium channel antagonist that has at least 20-fold selectivity for persistent sodium current relative to transient sodium current.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional and claims priority pursuant to 35U.S.C. § 120 to U.S. patent application Ser. No. 10/928,964, filed Aug.27, 2004, an application that claims priority pursuant to 35 U.S.C. §119(e) to provisional application Ser. No. 60/498,900 filed Aug. 29,2003, both of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the fields of neurobiology,physiology, biochemistry and medicine and can be directed toward thetreatment of pain and, in particular, to the therapeutic use ofcompounds that selectively reduce persistent sodium currents to treatchronic pain.

2. Background Information

The lipid bilayer membrane of all cells forms a barrier that is largelyimpermeable to the flux of ions and water. Residing within the membraneare a superfamily of proteins called ion channels, which provideselective pathways for ion flux. Precisely regulated conductancesproduced by ion channels are required for intercellular signaling andneuronal excitability. In particular, a group of ion channels that openupon depolarization of excitable cells are classified as voltage-gatedand are responsible for electrical activity in nerve, muscle and cardiactissue. In neurons, ion currents flowing through voltage-gated sodiumchannels are responsible for rapid spike-like action potentials. Duringaction potentials the majority of sodium channels open very briefly.These brief openings result in transient sodium currents. However, asubset of voltage-gated sodium channels does not close rapidly, butremain open for relatively long intervals. These channels thereforegenerate sustained or persistent sodium currents. The balance betweentransient and persistent sodium current is crucial for maintainingnormal physiological function and electrical signaling throughout theentire nervous system.

Clinical pain encompasses nociceptive and neuropathic pain. Each type ofpain is characterized by hypersensitivity at the site of damage and inadjacent normal tissue. While nociceptive pain usually is limited induration and responds well to available opioid therapy, neuropathic paincan persist long after the initiating event has healed, as is evident,for example, in phantom pain that often follows amputation. Chronic painsyndromes such as neuropathic pain can be triggered by a variety ofcauses, including, without limitation, a traumatic insult, such as,e.g., a compression injury, a spinal cord injury, a limb amputation, aninflammation or a surgical procedure; an ischemic event, such as, e.g.,a stroke; an infectious agent; a toxin exposure, such as, e.g., a drugor alcohol; or a disease such as, e.g., an inflammatory disorder, aneoplastic tumor, acquired immune deficiency syndrome (AIDS) or ametabolic disease.

Unfortunately, chronic pain such as chronic neuropathic pain isgenerally resistant to available opioid and nonsteroidalantiinflammatory drug therapies. Available drug treatments for chronicneuropathic pain, such as tricyclic antidepressants;anti-convulsants/anti-epileptic, such as, e.g., carbamazepine, phenyloinand lamotrigine; and local anesthetics/antiarrythmics, such as, e.g.,lidocaine, mexiletine, tocainide and flecainide, only temporarilyalleviate symptoms and to varying degrees. In addition, currenttherapies have serious side effects that can include cognitive changes,sedation, nausea, emesis, dizziness, ataxia, tinnitus and, in the caseof narcotic drugs, addiction. Further, many patients suffering fromneuropathic and other chronic pain are elderly or have medicalconditions that limit their tolerance to the side effects associatedwith available analgesic therapy, such as, e.g., cardiotoxicity, hepaticdysfunction and leukopenia. The inadequacy of current therapy inrelieving chronic pain without producing intolerable side effects isreflected in the high rate of depression and suicide in chronic painsufferers.

Recent evidence suggests that increased persistent sodium current may bean underlying basis for chronic pain, such as, e.g., inflammatory andneuropathic pain, see e.g., Fernando Cervero & Jennifer M. A. Laird,Role of Ion Channels in Mechanisms Controlling Gastrointestinal PainPathways, 3(6) CURR. OPIN. PHARMACOL. 608-612 (2003); Joel A. Black etal., Changes in the Expression of Tetrodotoxin-Sensitive Sodium ChannelsWithin Dorsal Root Ganglia Neurons in Inflammatory Pain, 108(3) PAIN237-247 (2004) and Li Yunru et al., Role of Persistent Sodium andCalcium Currents in Motoneuron Firing and Spasticity in Chronic SpinalRats, 91(2) J. NEUROPHYSIOL. 767-783 (2004), which are herebyincorporated by reference in their entirety. However, at present,treatments for chronic pain characterized by aberrant levels of sodiumchannel current, such as, e.g., Berger et al., Treatment of NeuropathicPain, U.S. Pat. No. 5,688,830 (Nov. 18, 1997); Marquess et al., SodiumChannel Drugs and Uses, U.S. Pat. No. 6,479,498 (Nov. 12, 2002); Choi etal., Sodium Channel Modulators, U.S. Pat. No. 6,646,012 (Nov. 11, 2003);and Chinn et al., Sodium Channel Modulators, U.S. Pat. No. 6,756,400(Jun. 29, 2004), encompass general sodium channel modulators that effecttransient currents. As such, the usefulness of available sodium channelblocking drugs is severely limited by potentially adverse side effects,such as, e.g., paralysis and cardiac arrest. Thus, there is a need fornovel methods of treating chronic pain that directly modulate persistentsodium current. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

The present invention provides methods of treating chronic pain in amammal, including a human. In one embodiment, the method involvesadministering to the mammal an effective amount of a selectivepersistent sodium current antagonist that has at least 20-foldselectivity for a persistent sodium current relative to transient sodiumcurrent. In further embodiments, the antagonist has at least 50-foldselectivity for a persistent sodium current, at least 200-foldselectivity for a persistent sodium current, at least 400-foldselectivity for a persistent sodium current, at least 600-foldselectively for a persistent sodium current, or at least 1000-foldselectively for a persistent sodium current, relative to a transientsodium current. A variety of mammals can be treated by the methods ofthe invention including, without limitation, humans.

The present invention provides methods of treating a variety of types ofchronic pain. In certain embodiments, the methods are directed totreating neuropathic pain, inflammatory pain such as arthritic pain,visceral pain, post-operative pain, pain resulting from cancer or cancertreatment, headache pain, irritable bowel syndrome pain, fibromyalgiapain, and pain resulting from diabetic neuropathy.

A variety of selective persistent sodium current antagonists can beuseful in the methods of the invention. In one embodiment, a method ofthe invention is practiced by administering an effective amount of aselective Na_(v)1.3 antagonist that has at least 20-fold selectivity forNa_(v)1.3 persistent sodium current relative to transient sodiumcurrent. In further embodiments, the antagonist has at least 50-foldselectivity for the Na_(v)1.3 persistent sodium current; at least200-fold selectivity for the Na_(v)1.3 persistent sodium current; atleast 400-fold selectivity for the Na_(v)1.3 persistent sodium current;at least 600-fold selectively for the Na_(v)1.3 persistent sodiumcurrent; or at least 1000-fold selectively for the Na_(v)1.3 persistentsodium current, relative to transient sodium current.

In further embodiments, the methods of the invention involveadministering an effective amount of a selective persistent sodiumcurrent antagonist belonging to one of the disclosed structural classesof selective persistent sodium current antagonists. Such a selectivepersistent sodium channel antagonist can be, without limitation, acompound represented by a formula selected from Formula 1:

wherein,

Ar¹ is an aryl group;

Ar² is an aryl group;

Y is absent or is selected from:

R¹ is selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl;

R² and R³ are independently selected from hydrogen, C₁-C₈ alkyl, aryl,arylalkyl, hydroxy, fluoro, C₁-C₈ carbocyclic ring, or C₁-C₈heterocyclic ring;

R⁴ is selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl; and

n is an integer of from 1 to 6;

wherein,

Ar³ is an aryl group;

Ar⁴ is an aryl group;

X¹ and Y¹ are independently selected from

R⁵ and R⁶ are independently selected from hydrogen, fluoro, C₁ to C₈alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R⁸ and R⁹ are selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl,COR¹², COCF₃;

R¹⁰ and R¹¹ are selected from hydrogen, halogen, hydroxyl, C₁-C₈ alkyl,aryl, arylalkyl, and

R¹² is selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl;

wherein,

Ar⁵ is an aryl group;

Ar⁶ is an aryl group;

X² is O, S, or NR¹⁴;

Y² is N or CR¹⁵;

Z² is N or CR¹⁶;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R¹³ is selected from halogen, C₁-C₈ alkyl, arylalkyl, and(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁴ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁵ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁶ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂; and

c is 0 or an integer from 1 to 5; and

wherein,

Ar⁷ is an aryl group;

R is selected from halogen, C₁-C₈ alkyl, NR²²R²³, OR²²;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R¹⁷ and R¹³ are independently selected hydrogen, C₁-C₈ alkyl, aryl,arylalkyl, hydroxy;

R¹⁹ and R²⁰ are independently selected from hydrogen, halogen, C₁-C₈alkyl, hydroxy, amino, CF₃;

R²¹, R²², and R²³ are independently selected from hydrogen, aryl orC₁-C₈ alkyl;

a is 0 or an integer from 1 to 5; and

m is 0 or and integer from 1 to 3.

A compound corresponding to any of the above formulas also can be apharmaceutically acceptable salt, ester, amide, or geometric,steroisomer, or racemic mixture.

Any of the variety of routes of administration can be useful fortreating chemical pain according to a method of the invention. Inparticular embodiments, administration is performed peripherally,systemically or orally.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows four compounds that are selective persistent sodium currentantagonists.

FIG. 2 shows inhibition of persistent current-dependent depolarizationby sodium channel blockers. In the this assay, cells are resting inwells containing 80 μl of TEA-MeSO₃ (sodium-free box) to which is added240 μl of NaMeSO₃ buffer containing 13 mM KMeSO₃ for a final K⁺concentration of 10 mM and a final Na⁺ concentration of 110 mM(sodium/potassium-addition). This elicits a robust depolarizingresponse. Following the resolution of the sodium-dependentdepolarization, a second aliquot of KMeSO₃ is added to the well bringingthe final K⁺ concentration to 80 mM (High potassium-addition). Thisaddition results in a second depolarizing response. Compounds thatreduce the sodium-dependent, but not the potassium-dependentdepolarizations are selected as persistent sodium channel blockers.Circles indicate the control response with 0.1% DMSO added, trianglesshow the effects of the sodium channel inhibitor tetracaine (10 μM) andthe diamonds show the response during the application of a non-specificchannel blocker.

FIG. 3 shows data from assays in which the screening window for thepersistent current assay is determined. To evaluate the size of the“screening window,” data was examined from assays in which responses tosodium-dependent depolarization were measured in the presence of 10 μMTetracaine to completely block the sodium-dependent depolarization or inthe presence of a 0.1% DMSO control to obtain a maximum depolarization.Data were binned into histograms and a screening window (Z) wascalculated from the mean and standard deviation for the maximal andminimum values according to the equation:Z=1−(3×STD_(Max)+3×STD_(Min))/(Mean_(Max)−Mean_(Min)). Histograms A, Band C represent data obtained from three different assay plates. Thescreening window for a run was considered adequate 1.0≧Z≧0.5.

FIG. 4 shows sodium current traces before and after the addition of 3 μMCompound 1 or 500 nM TTX. HEK cells expressing Na_(v)1.3 channels werepatch clamped in the perforated-patch mode. Currents were elicited by200 msec test pulses to 0 mV from a holding potential of −90 mV.

FIG. 5 shows a dose-response curve for Compound 1. The peak amplitudesof transient Na⁺ current (I_(t)) and the steady state amplitude of thepersistent current (I_(p)) were measured at various Compound 1concentrations, normalized to amplitude of the control currents. Thepercent block was then plotted against drug concentration. Solid linesrepresent fits to the data with the Hill equation. The calculated EC₅₀values and Hill coefficients are as follows: Hillslope, I_(t) is 0.354and I_(p) is 0.733; EC₅₀, I_(t) is 0.167 M and I_(p) is 3.71×10⁻⁶ M.

FIG. 6 shows the effects of intraperitoneally administered Compound 1 onpaw withdrawal threshold (mean±SEM) in a test of mechanical allodynia inthe spinal nerve ligation model of neuropathic pain. Paw withdrawalthreshold (gram force) was determined using von Frey filamentstimulation and the Dixon's up-down method. Allodynic response wasmeasured at baseline (0 min) and at 15, 30, 60 and 120 min after of 10mg/kg IP injection of Compound 1 or vehicle control. Percent reversal ofallodynia compared with non-injected rats was calculated. Six rats wereused at each dose. Data were analyzed by analysis of variance andDunnett's test reversal of allodynia was considered significant ifP<0.05

DETAILED DESCRIPTION OF THE INVENTION

I. Voltage-Gated Sodium Channels

In the normal functioning of the nervous system, neurons are capable ofreceiving a stimulus, and in response, propagating an electrical signalaway from their neuron cell bodies (soma) along processes (axons). Fromthe axon, the signal is delivered to the synaptic terminal, where it istransferred to an adjacent neuron or other cell. Voltage-sensitivesodium channels have an important role in nervous system functionbecause they mediate propagation of electrical signals along axons.

Voltage-gated sodium channels are members of a large mammalian genefamily encoding at least 9 alpha- and 3 beta-subunits. While all membersof this family conduct Na⁺ ions through the cell membrane, they differin tissue localization, regulation and, at least in part, in kinetics ofactivation and inactivation, see, e.g., William A. Catterall, From IonicCurrents to Molecular Mechanism: The Structure and Function ofVoltage-gated Sodium Channels, 26(1) NEURON 13-25 (2000); and Sanja D.Novakovic et al., Regulation of Na ⁺ Channel Distribution in the NervousSystem, 24(8) TRENDS NEUROSCI. 473-478 (2001), which are herebyincorporated by reference in their entirety.

Generally, under resting conditions, sodium channels are closed until astimulus depolarizes the cell to a threshold level. At this threshold,sodium channels begin to open and then rapidly generate the upstroke ofthe action potential. Normally during an action potential, sodiumchannels open briefly (one millisecond) and then close (inactivate)until the excitable cell returns to its resting potential and the sodiumchannels re-enter the resting state.

Without wishing to be bound by the following, this behavior ofvoltage-gated sodium channels can be understood as follows. Sodiumchannels can reside in three major conformations or states. The restingor “closed” state predominates at negative membrane potentials (≦−60mV). Upon depolarization, channels open and allow current to flow.Transition from the resting state to the open state occurs within amillisecond after depolarization to positive membrane potentials.Finally, during sustained depolarization (>1-2 ms), channels enter asecond closed or inactive state. Subsequent re-opening of channelsrequires recycling of channels from an inactive state to a restingstate, which occurs when the membrane potential returns to a negativevalue (repolarization). Therefore, membrane depolarization not onlycauses sodium channels to open, but also causes them to close, duringsustained depolarization.

A small fraction of the sodium channels can fail to inactivate even withsustained depolarization. This non-inactivating sodium current is calleda “persistent” sodium current. Four sodium channels, Nav1.3, Nav1.5,Nav1.6 and Nav1.9, have historically been known to generate a persistentcurrent. Recent evidence, however, suggests that all voltage-gatedsodium channels are capable of producing a persistent current, see,e.g., Abraha Taddese & Bruce P. Bean, Subthreshold Sodium Current fromRapidly Inactivating Sodium Channels Drives Spontaneous Firing ofTubermammillary N EURONS, 33(4) NEURON 587-600 (2002); Michael Tri H. Do& Bruce P. Bean, Subthreshold Sodium Currents and Pacemaking ofSubthalamic Neurons: Modulation by Slow Inactivation, 39(1) NEURON109-120 (2003), which are hereby incorporated by reference in theirentirety. The mechanism that produces a persistent current is poorlyunderstood. Two hypothesis are (1) that different sodium channelsproduce transient and persistent currents, and (2) that a sodium channelcapable of producing transient sodium current enters a different gatingmode to produce a persistent current. Persistent sodium channels canopen at more negative membrane potentials relative to normal sodiumchannels and inactivate at more positive potentials, see, e.g., JacopoMagistretti & Angel Alonso, Biophysical Properties and Slow-voltageDependent Inactivation of a Sustained Sodium Current in EntorhinalCortex Layer-II Principal Neurons: A Whole-Cell and Single-Channel Study114(4) J. GEN. PHYSIOL. 491-509 (1999). Persistent sodium current havebeen observed at membrane potentials as negative as −80 mV, see, e.g.,Peter K. Stys, Anoxic and Ischemic Injury of Myelinated Axons in CNSWhite Matter: From Mechanistic Concepts to Therapeutics, 18(1) J. CEREB.BLOOD FLOW METAB. 2-25 (1998) and have been shown to persist for secondsfollowing depolarization at potentials as positive as 0 mV, see, e.g.,Magistretti & Alonso, supra, (1999). Thus, persistent sodium current isdistinct from, and can be readily distinguished from, transient sodiumcurrent.

Although the physiological role of persistent sodium current is notfully understood, such current can function in generating rhythmicoscillations; integrating synaptic input; modulating the firing patternof neurons; and regulating neuronal excitability and firing frequency,see, e.g., Wayne E. Crill, Persistent Sodium Current in MammalianCentral Neurons 58 ANNU. REV. PHYSIOL. 349-362 (1996); and David S.Ragsdale & Massimo Avoli, Sodium Channels as Molecular Targets forAntiepileptic Drugs, 26(1) BRAIN RES. BRAIN RES. REV. 16-28 (1998).Persistent sodium current also can induce deleterious phenomena,including cardiac arrhythmia, epileptic seizure, and neuronal cell deathunder ischemic and anoxic conditions, see, e.g., Christoph Lossin etal., Molecular Basis of an Inherited Epilepsy 34(6) NEURON 877-84(2002); Peter K. Stys et al., Ionic Mechanisms of Anoxic Injury inMammalian CNS White Matter: Role of Na ⁺ Channels and Na(+)-Ca2+Exchanger, 12(2) J. NEUROSCI. 430-439 (1992); Peter K. Stys et al.,Noninactivating, Tetrodotoxin-Sensitive Na ⁺ Conductance in Rat OpticNerve Axons, 90(15) PROC. NATL. ACAD. SCI. USA, 6976-6980 (1993); andGiti Garthwaite et al., Mechanisms of Ischaemic Damage to Central WhiteMatter Axons: A Quantitative Histological Analysis Using Rat OpticNerve, 94(4) NEUROSCIENCE 1219-1230 (1999). Thus, aberrant persistentsodium current can contribute to development or progression ofpathological conditions by collapsing the normal cell transmembranegradient for sodium, leading to reverse operation of the sodium-calciumexchanger, and resulting in an influx of intracellular calcium, whichinjures the axon, see, e.g., Stys et al., supra, (1992).

While abnormal activity of a persistent current can underlie a widearray of chronic pain conditions, the underlying mechanisms appears tobe similar. It is generally understood that abnormally increasedpersistent sodium current can depolarize the resting membrane potentialor reduce the rate of repolarization during an action potential. Eithereffect may produce a state of hyper-excitability in which aberrantneuronal behavior is manifested. This aberrant neuronal behavior canresult in a neuron with increased firing rates, enhanced sensitivity tosynaptic input or abnormal repetitive or rhythmic firing patterns. It isalso generally understood that abnormally high levels of persistentcurrent generate sustained membrane depolarization and a concomitantincrease of Na⁺ within the cell. This high Na⁺ influx, in turn, drivesthe sodium/calcium exchanger, which in turn, results in detrimentallevels of Ca²⁺ to accumulate inside affected cells. Abnormally highlevels of Ca²⁺ result in neuronal cell dysfunction and neuronal celldeath. Thus, by collapsing the normal cell transmembrane gradient forsodium, a persistent current can reverse the operation of thesodium-calcium exchanger, and the resulting an influx of intracellularcalcium would cause injures or death to a nerve. As disclosed herein,conditions associated with aberrant persistent sodium current can betreated by selectively inhibiting or reducing persistent sodium currentwithout compromising normal transient sodium current function, therebyallowing normal neuronal function (excitability). As disclosed herein,pain conditions associated with aberrant persistent sodium current canbe treated by selectively inhibiting or reducing persistent sodiumcurrent without compromising normal transient sodium current function.

II. Chronic Pain and Persistent Sodium Current

There is strong evidence that altered voltage-gated sodium channelactivity plays a critical role in chronic pain, such as, e.g.,inflammatory and neuropathic pain, see, e.g., Mark D. Baker & John N.Wood, Involvement of Na ⁺ Channels in Pain Pathways, 22(1) TRENDSPHARMACOL. SCI. 27-31 (2001); John N. Wood et al., Sodium Channels inPrimary Sensory Neurons: Relationship to Pain States, 241 NOVARTISFOUND. SYMP. 159-168 (2002); Josephine Lai et al., The Role ofVoltage-gated Sodium Channels in Neuropathic Pain, 13(3) CURR. OPIN.NEUROBIOL. 291-297 (2003); Philip LoGrasso & Jeffrey McKelvy, Advancesin Pain Therapeutics, 7(4) CURR. OPIN. CHEM. BIOL. 452-456 (2003);Phillip J. Birch et al., Strategies to Identify Ion Channel Modulators:Current and Novel Approaches to Target Neuropathic Pain, 9(9) DRUGDISCOV. TODAY 410-418 (2004); and Josephine Lai et al., Voltage-gatedsodium channels and hyperalgesia, 44 ANNU. REV. PHARMACOL. TOXICOL.371-397 (2004), which are hereby incorporated by reference in theirentirety. Alterations in sodium channel expression and/or function has aprofound effect on the firing pattern of neurons in both the peripheraland central nervous systems. For example, injury to sensory primaryafferent neurons often results in rapid redistribution of voltage-gatedsodium channels along the axon and dendrites and in abnormal, repetitivedischarges or exaggerated responses to subsequent sensory stimuli. Suchan exaggerated response is considered to be crucial for the incidence ofspontaneous pain in the absence of external stimuli that ischaracteristic of chronic pain. In addition, inflammatory pain isassociated with lowered thresholds of activation of nociceptors in theperiphery and altered sodium channel function is thought to underlieaspects of this phenomenon. Likewise, neuropathic pain states resultingfrom peripheral nerve damage is associated with altered sodium channelactivity and ectopic action potential propagation.

Importantly, sodium channel inhibitors are clinically effective in thetreatment of many types of chronic pain. For example, local anesthetics(such as, e.g., lidocaine, mexiletine, tocainide and flecainide) havebeen reported to provide effective relief in painful diabeticneuropathy, neuralgic pain, lumbar radiculopathies, complex regionalpain syndrome Type I and Type II and traumatic peripheral injuries.Anticonvulsants (such as, e.g., carbamazepine and phenyloin) used asanalgesics to treat chronic pain associated with neuralgic pain,trigeminal neuralgia, diabetic neuropathy. Anti-epileptic agents (suchas, e.g., lamotrigine) are used with trigeminal neuralgia, diabeticneuropathy, postherpetic neuralgia, complex regional pain syndrome TypeII and phantom pain. However, the usefulness of available sodium channelblocking drugs is severely limited by their failure to discriminateadequately between sodium channel a subunits. Highly systemicconcentration would be associated with devastating side-effects, suchas, e.g., periodic paralyses in muscle, cardiac arrest due toventricular fibrillation and delayed cardiac repolarization in theheart, and epilepsy in the central nervous system, see, e.g., Baker &Wood, supra, (2001); and Lai et al., supra, (2004).

Recent evidence has revealed that increased activity from a persistentsodium current may be responsible for the underlying basis of chronicpain, see e.g., Cervero & Laird, supra, (2003); Black et al., supra,(2004); and Yunru et al., supra, (2004), which are hereby incorporatedby reference in their entirety. An example of a sodium channel capableof mediating persistent current is the type III sodium channelNa_(v)1.3. Under pathological pain circumstances, Na_(v)1.3 expressioncan become upregulated while other sodium channels are concomitantlydownregulated. For example, in adult rodents, damage to sensory neuronsresults in upregulation of Na_(v)1.3 and downregulation of Na_(v)1.8 andNa_(v)1.9, see, e.g., Birch et al., supra, (2004), which is herebyincorporated by reference in its entirety. Furthermore, this Na_(v)1.3upregulation after nerve injury is associated with increased membranepotential oscillations that appear to underlie spontaneous activity,see, e.g., Bryan C. Hains et al., Upregulation of Sodium Channel Na_(v)1.3 and Functional Involvement in Neuronal HyperexcitabilityAssociated With Central Neuropathic Pain After Spinal Cord Injury,23(26) J. NEUROSCI. 8881-8892 (2003); and Bryan C. Hains et al., AlteredSodium Channel Expression in Second-Order Spinal Sensory NeuronsContributes to Pain after Peripheral Nerve Injury, 24(20) J. NEUROSCI.4832-4839 (2004), which are hereby incorporated by reference in theirentirety. Selective reduction in the expression or activity of sodiumchannels capable of mediating persistent current relative to anyreduction in normal voltage-gated (transient) sodium current can beuseful for treating conditions associated with increased persistentsodium current.

Therefore, chronic pain is an example of a condition associated withincreased persistent sodium current. As described herein, a compoundthat decreases persistent sodium current without a similar decrease innormal transient sodium current can effectively treat chronic painwithout harmful side effects that generally accompany non-selectivesodium channel blockers. As disclosed in Example 4, a selectivepersistent sodium current antagonist can effectively reverse allodyniain an animal model of neuropathic pain. Therefore, based on theidentification of selective persistent sodium channel antagonists thathave at least 20-fold selectivity for persistent sodium channel relativeto transient sodium current, and the demonstration of the effectivenessof treating pain by selectively antagonizing persistent sodium current,the present invention provides a method of treating chronic pain in amammal by selectively antagonizing persistent sodium current. The methodinvolves administering to the mammal an effective amount of a selectivepersistent sodium channel antagonist that has at least 20-foldselectivity for persistent sodium current relative to transient sodiumcurrent.

The methods of the invention are useful for treating any of a variety oftypes of chronic pain, and, as non-limiting examples, pain that isneuropathic, visceral or inflammatory in origin. In particularembodiments, the methods of the invention are used to treat neuropathicpain; visceral pain; post-operative pain; pain resulting from cancer orcancer treatment; fibromyalgia pain, and inflammatory pain.

As used herein, the term “pain” encompasses both acute and chronic pain.As used herein, the term “acute pain” means immediate, generally highthreshold, pain brought about by injury such as a cut, crush, burn, orby chemical stimulation such as that experienced upon exposure tocapsaicin, the active ingredient in chili peppers. The term “chronicpain,” as used herein, means pain other than acute pain and includes,without limitation, neuropathic pain, visceral pain, inflammatory pain,headache pain, muscle pain and referred pain. It is understood thatchronic pain often is of relatively long duration, for example, monthsor years and can be continuous or intermittent.

In one embodiment, the methods of the invention are used to treat“neuropathic pain,” which, as used herein, means abnormal sensory inputby either the peripheral nervous system, central nervous systems, orboth resulting in discomfort. Neuropathic pain typically is long-lastingor chronic and can develop days or months following an initial acutetissue injury. Symptoms of neuropathic pain can involve persistent,spontaneous pain, as well as allodynia, which is a painful response to astimulus that normally is not painful, hyperalgesia, an accentuatedresponse to a painful stimulus that usually a mild discomfort, such as apin prick, or hyperpathia, a short discomfort becomes a prolonged severepain. Neuropathic pain generally is resistant to opioid therapy.Neuropathic pain can be distinguished from nociceptive pain, which ispain caused by the normal processing of stimuli resulting from acutetissue injury. In contrast to neuropathic pain, nociceptive pain usuallyis limited in duration to the period of tissue repair and usually can bealleviated by available opioid and non-opioid analgesics.

The methods of the invention are useful for treating bothcentrally-generated and peripherially-generated neuropathic painresulting from, without limitation, a trauma or disease of peripheralnerve, dorsal root ganglia, spinal cord, brainstem, thalamus or cortex.Examples of neuropathic pain that can be treated by the methods of theinvention include neuralgia, such as, e.g., trigeminal neuralgia,post-herpetic neuralgia, glossopharyngeal neuralgia, sciatica andatypical facial pain; deafferentation pain syndromes, such as, e.g.,injury to the brain or spinal cord, post-stroke pain, phantom pain,paraplegia, peripheral nerve injuries, brachial plexus avulsioninjuries, lumbar radiculopathies and postherpetic neuralgia; complexregional pain syndromes (CRPSs) such as, e.g., reflex sympatheticdystrophy (CRPS Type I) and causalgia (CRPS Type II); andpolyneuropathic pain, such as, e.g., diabetic neuropathy,chemotherapy-induced pain, treatment-induced pain, and postmastectomysyndrome. It is understood that the methods of the invention are usefulin treating neuropathic pain regardless of the etiology of the pain. Asnon-limiting examples, the methods of the invention can be used to treatneuropathic pain resulting from a peripheral nerve disorder such asneuroma; from nerve compression; from nerve crush or stretch, nerveentrapment or incomplete nerve transsection; or from a mononeuropathy ora polyneuropathy. As further non-limiting examples, the methods of theinvention are useful in treating neuropathic pain resulting from adisorder such as dorsal root ganglion compression; inflammation of thespinal cord; contusion, tumor or hemisection of the spinal cord; andtumors or trauma of the brainstem, thalamus or cortex.

As indicated above, the methods of the invention can be useful fortreating neuropathic pain resulting from a mononeuropathy,polyneuropathy, complex regional pain syndromes or deafferentation. Aneuropathy is a functional disturbance or pathological change in theperipheral nervous system and is characterized clinically by sensory ormotor neuron abnormalities. The term mononeuropathy indicates that asingle peripheral nerve is affected, while the term polyneuropathyindicates that several peripheral nerves are affected. Deafferentationindicates a loss of the sensory input from a portion of the body, andcan be caused by interruption of either peripheral sensory fibres ornerves from the central nervous system. The etiology of a neuropathy canbe known or unknown. Known etiologies include complications of a diseaseor toxic state such as diabetes, which is the most common metabolicdisorder causing neuropathy, or irradiation, ischemia or vasculitis.Polyneuropathies that can be treated by a method of the invention canresult, without limitation, from post-polio syndrome, diabeticneuropathy, alcohol neuropathy, amyloid, toxins, AIDS, hypothyroidism,uremia, vitamin deficiencies, chemotherapy, 2′,3′-didexoycytidine (ddC)treatment, Guillain-Barre syndrome or Fabry's disease. It is understoodthat the methods of the invention can be used to treat chronic pain ofthese or other chronic neuropathies of known or unknown etiology.

The methods of the invention also can used for treating chronic painresulting from excessive muscle or nerve tension, such as certain typesof back pain, such as that resulting from a herniated disc; a bone spur,sciatica, sprains, strains and joint pain. The methods of the inventioncan further be used for treating chronic pain resulting from activity,such as, as non-limiting examples, long hours of work at a computer,work with heavy objects or heavy machinery, or spending long hours onone's feet, and repetitive motion disorders (RMDs). RMDs are a varietyof muscular conditions that can cause chronic pain. RMDs can be causedby overexertion, incorrect posture, muscle fatigue, compression ofnerves or tissue, too many uninterrupted repetitions of an activity ormotion, or friction caused by an unnatural or awkward motion such astwisting the arm or wrist. Common RMDs occur in the hands, wrists,elbows, shoulders, neck, back, hips, knees, feet, legs, and ankles,however, the hands and arms are most often affected. The methods of theinvention can be used to treat chronic pain arising from any type ofRMD. The methods of the invention further can be used to treat chronicmuscle pain, chronic pain associated with substance abuse or withdrawal,and other types of chronic pain of known or unknown etiology.

Similarly, the methods of the invention can be used to treat chronicpain resulting from an inflammatory disorder, for example, fromarthritis/connective tissue disorders such as, e.g., osteoarthritis,rheumatoid arthritis, juvenile arthritis, gouty arthritis;spondyloarthritis, scleroderma and fibromyalgia; autoimmune diseasessuch as, e.g., Guillain-Barre syndrome, myasthenia gravis and lupuserythematosus; inflammation caused by injury, such as a crush, puncture,stretch of a tissue or joint; inflammation caused by infection, such astuberculosis; or neurogenic inflammation.

The methods of the invention can also be used to treat visceral pain,such as, e.g., functional visceral pain including chronicgastrointestinal inflammations like Crohn's disease, ulcerative colitis,gastritis, irritable bowel syndrome; orangic visceral pain includingpain resulting from a traumatic, inflammatory or degenerative lesion ofthe gut or produced by a tumor impinging on sensory innervation; andtreatment-induced visceral pain, for example, attendant to chemotherapyor radiation therapy.

The methods of the invention can be used for treating chronic painresulting from headache, including, without limitation, tension-typeheadache, migraine headache, cluster headache, hormone headache, reboundheadache, sinus headache, and organic headache. The methods of theinvention can be used for treating chronic pain resulting infections,such as, e.g., Lymes disease, HIV/AIDS and leprosy.

III. Selective Persistent Sodium Current Blockers

The methods of the invention involve administering a compound thatselectively reduces persistent sodium current relative to transientsodium current. As used herein, the term “selective,” when used hereinin reference to a compound, such as an antagonist, means a compoundthat, at least one particular dose reduces persistent sodium current atleast 20-fold more than transient sodium current is reduced. Therefore,a compound that selectively reduces persistent sodium current has atleast 20-fold selectively for persistent sodium current relative totransient sodium current, and can have, for example, at least 50-foldselectively for persistent sodium current relative to transient sodiumcurrent, at least 100-fold, at least 200-fold, at least 400-fold, atleast 600-fold, or at least 1000-fold selectively for persistent sodiumcurrent relative to transient sodium current.

As used herein, the term “persistent sodium current” means a sodiumchannel mediated current that is non-transient; that can remain activeduring prolonged depolarization or that activates at voltage morenegative than −60 mV and thus can contribute to hyperexcitability of theneural membrane. Prolonged depolarization refers to depolarization thatoccurs over a time period greater than the time period during which atransient current typically inactivates. As a non-limiting example,prolonged depolarization can occur within a time period greater than thetime period during which the transient current of a sodium channel, suchas Na_(v)1.2, typically inactivates. Therefore, prolonged depolarizationrefers to depolarization that persists for at least 0.002 second, suchas at least 0.01 second, at least 0.1 second and at least 1 second.

A compound that selectively reduces persistent sodium current can be,for example, a persistent sodium channel antagonist. As used herein, theterm “persistent sodium channel antagonist,” means a compound thatinhibits or decreases persistent current mediated through a sodiumchannel by binding to the sodium channel. It is understood that apersistent sodium channel antagonist can act by any antagonisticmechanism, such as by directly binding a persistent sodium channel atthe pore entrance, thereby inhibiting movement of ions through thechannel, or by binding a channel at another site to alter channelconformation and inhibit movement of ions through the channel. Exemplaryselective persistent sodium channel antagonists that represent fourstructural classes of organic molecules are disclosed herein as Formulas1, 2, 3 and 4.

It further is understood that a compound that selectively reducespersistent sodium current can act indirectly, for example, by reducingor down-regulating expression of a persistent sodium channel, forexample, by inactivating a positive regulator of transcription oractivating a negative regulator of transcription, without acorresponding reduction transient sodium channel; by increasing theexpression or activity of a molecule that inactivates or reducespersistent sodium channel function, such as a protease, modifying enzymeor other molecule, without a corresponding reduction in transient sodiumcurrent; or by decreasing the expression or activity of a molecule thattransmits a downstream signal from a persistent sodium current without acorresponding reduction in transient sodium current, for example,without substantially altering the downstream signal from a transientsodium channel.

As disclosed herein, structurally unrelated molecules can have at least20-fold selectivity for reducing persistent sodium current relative totransient sodium current and, therefore, can be useful in the methods ofthe invention. For example, such a compound can be a naturally ornon-naturally occurring macromolecule, such as a peptide,peptidomimetic, nucleic acid, carbohydrate or lipid. The compoundfurther can be an antibody, or antigen-binding fragment thereof such asa monoclonal antibody, humanized antibody, chimeric antibody, minibody,bifunctional antibody, single chain antibody (scFv), variable regionfragment (Fv or Fd), Fab or F(ab)₂. The compound also can be a partiallyor completely synthetic derivative, analog or mimetic of a naturallyoccurring macromolecule, or a small organic or inorganic molecule.

A selective persistent sodium current antagonist that is a nucleic acidcan be, for example, an anti-sense nucleotide sequence, an RNA molecule,or an aptamer sequence. An anti-sense nucleotide sequence can bind to anucleotide sequence within a cell and modulate the level of expressionof a persistent sodium channel gene, or modulate expression of anothergene that controls the expression or activity of a persistent sodiumchannel. Similarly, an RNA molecule, such as a catalytic ribozyme, canbind to and alter the expression of a persistent sodium channel gene, orother gene that controls the expression or activity of a persistentsodium channel. An aptamer is a nucleic acid sequence that has a threedimensional structure capable of binding to a molecular target, see,e.g., Sumedha D. Jayasena, Aptamers: An Emerging Class of Molecules ThatRival Antibodies in Diagnostics, 45(9) CLIN. CHEM. 1628-1650 (1999),which is hereby incorporated by reference in its entirety. As such, anaptamer can serve as a persistent sodium current selective compound.

A selective persistent sodium current antagonist that is a nucleic acidalso can be a double-stranded RNA molecule for use in RNA interferencemethods. RNA interference (RNAi) is a process of sequence-specific genesilencing by post-transcriptional RNA degradation, which is initiated bydouble-stranded RNA (dsRNA) homologous in sequence to the silenced gene.A suitable double-stranded RNA (dsRNA) for RNAi contains sense andantisense strands of about 21 contiguous nucleotides corresponding tothe gene to be targeted that form 19 RNA base pairs, leaving overhangsof two nucleotides at each 3′ end (Sayda M. Elbashir et al., Duplexes of21-nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells,411(6836) NATURE 494-498 (2001); B. L. Bass, RNA Interference. The ShortAnswer, 411(6836) NATURE 428-429 (2001); Phillip D. Zamore, RNAInterference: Listening to the Sound of Silence, 8(9) NAT. STRUCT. BIOL.746-750 (2001), which are hereby incorporated by reference in theirentirety. dsRNAs of about 25-30 nucleotides have also been usedsuccessfully for RNAi (Anton Karabinos et al., Essential Roles for FourCytoplasmic Intermediate Filament Proteins in Caenorhabditis elegansDevelopment, 98(14) PROC. NATL. ACAD. SCI. USA 7863-7868 (2001), whichis hereby incorporated by reference in its entirety. dsRNA can besynthesized in vitro and introduced into a cell by methods known in theart.

A persistent sodium channel selective compound that is an antibody canbe, for example, an antibody that binds to a persistent sodium channeland inhibits movement of ions through the channel, or alters theactivity of a molecule that regulates persistent sodium currentexpression or activity, such that sodium current is decreased. It isunderstood that such a compound binds selectively such that acorresponding reduction in transient sodium current is not affected.

A persistent sodium channel selective compound that is a small moleculecan have a variety of structures. In several embodiments, a compoundthat selectively reduces persistent sodium current that has at least20-fold selectivity for reducing persistent sodium current tonon-persistent sodium current is an organic molecule represented by aformula shown herein below, or a pharmaceutically acceptable salt,ester, amide, steroisomer or racemic mixture thereof. As disclosedherein in FIG. 1, several identified compounds are selective forpersistent sodium current relative to transient sodium current, withselectivities of 32-fold, 38-fold, 110-fold and 453-fold. It isunderstood that these and other compounds with at least 20-foldselectivity for persistent sodium current relative to transient sodiumcurrent, for example, identified by the methods disclosed herein inExamples 1, 2, 3 and 4 can be useful for treating chronic pain accordingto a method of the invention.

In one embodiment, a compound useful in a method of the invention, or apharmaceutically acceptable salt, ester, amide, stereoisomer or racemicmixture thereof, has a structure from Formula 1:

wherein,

Ar¹ is an aryl group;

Ar² is an aryl group;

Y is absent or is selected from:

R¹ is selected from hydrogen, C₁-C₈ alkyl, aryl, or arylalkyl;

R² and R³ are independently selected from hydrogen, C₁-C₈ alkyl, aryl,arylalkyl, hydroxy, fluoro, C₁-C₈ carbocyclic ring, or C₁-C₈heterocyclic ring;

R⁴ is selected from hydrogen, C₁-C₈ alkyl, aryl, or arylalkyl;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, orhydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, or arylalkyl, and

n is an integer of from 1 to 6.

In one aspect of this embodiment, Ar¹ is thienyl, or substitutedthienyl. For example, the thienyl can be substituted with one or more ofhalogen, C₁-C₈ alkyl, NO₂, CF₃, OCF₃, OCF₂H, CN, (CR⁵R⁶)_(c)N(R⁷)₂,wherein c is 0 or an integer from 1 to 5; and

In another aspect of this embodiment, Ar² is phenyl or substitutedphenyl. For example, the phenyl can be substituted with halogen, C₁-C₈alkyl, arylalkyl, NO₂, CF₃, OCF₃, OCF₂H, CN and (CR⁵R⁶)_(c)N(R⁷)₂,wherein c is 0 or an integer from 1 to 5.

In another embodiment, a compound useful in a method of the invention,or a pharmaceutically acceptable salt, ester, amide, stereoisomer orracemic mixture thereof, has a structure from Formula 2:

wherein,

Ar³ is an aryl group;

Ar⁴ is an aryl group;

X¹ and Y¹ are independently selected from:

R⁵ and R⁶ are independently selected from: hydrogen, fluoro, C₁ to C₈alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R⁸ and R⁹ are selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl,COR¹², COCF₃;

R¹⁰ and R¹¹ are selected from hydrogen, halogen, hydroxyl, C₁-C₈ alkyl,aryl, arylalkyl; and

R¹² is selected from hydrogen, C₁-C₈ alkyl, aryl, arylalkyl.

In one aspect of this embodiment, Ar³ can be phenyl or substitutedphenyl. For example, the phenyl can be substituted with one or more ofhalogen, C₁-C₈ alkyl, NO₂, CF₃, OCF₃, OCF₂H, CN, (CR⁵R⁶)_(c)N(R⁷)₂,wherein c is 0 or an integer from 1 to 5.

In another aspect of this embodiment, Ar⁴ is substituted with one ormore of halogen, C₁-C₈ alkyl, arylalkyl, NO₂, CF₃, OCF₃, OCF₂H, CN or(CR⁵R⁶)_(c)N(R⁷)₂, wherein c is 0 or an integer from 1 to 5.

In yet another embodiment, a compound useful in a method of theinvention, or a pharmaceutically acceptable salt, ester, amide,stereoisomer or racemic mixture thereof, has a structure from Formula 3:

wherein,

Ar⁵ is an aryl group;

Ar⁶ is an aryl group;

X² is O, S, or NR¹⁴;

Y² is N or CR¹⁵;

Z² is N or CR¹⁶;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R¹³ is selected from halogen, C₁-C₈ alkyl, arylalkyl, and(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁴ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁵ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂;

R¹⁶ is selected from hydrogen, halogen, C₁ to C₈ alkyl, CF₃, OCH₃, NO₂,(CR⁵R⁶)_(c)N(R⁷)₂, and

wherein c is 0 or an integer from 1 to 5.

In one aspect of this embodiment, Ar⁵ is phenyl or substituted phenyl.For example, the phenyl can be substituted with one or more of halogen,C₁-C₈ alkyl, NO₂, CF₃, OCF₃, OCF₂H, CN, or (CR⁵R⁶)_(c)N(R⁷)₂, wherein cis 0 or an integer from 1 to 5.

In another aspect of this embodiment, Ar⁶ is substituted with halogen,C₁-C₈ alkyl, arylalkyl, NO₂, CF₃, OCF₃, OCF₂H, CN or (CR⁵R⁶)_(c)N(R⁷)₂wherein c is 0 or an integer from 1 to 5.

In yet another aspect of this embodiment, Ar⁶ is selected from:

In yet another embodiment, a compound useful in a method of theinvention, or a pharmaceutically acceptable salt, ester, amide,stereoisomer or racemic mixture thereof, has a structure from Formula 4:

wherein,

Ar⁷ is an aryl group;

R_(a) is selected from halogen, C₁-C₈ alkyl, NR²²R²³, OR²²;

R⁵ and R⁶ are selected from hydrogen, fluoro, C₁ to C₈ alkyl, hydroxy;

R⁷ is selected from hydrogen, C₁ to C₈ alkyl, aryl, arylalkyl;

R¹⁷ and R¹ are independently selected hydrogen, C₁-C₈ alkyl, aryl,arylalkyl, and hydroxy;

R¹⁹ and R²⁰ are independently selected from hydrogen, halogen, C₁-C₈alkyl, hydroxy, amino, CF₃;

R²¹, R²², and R²³ are independently selected from hydrogen, aryl orC₁-C₈ alkyl;

a is 0 or an integer from 1 to 5, and

m is 0 or and integer from 1 to 3.

In one aspect of this embodiment, Ar⁷ is phenyl or substituted phenyl.For example the phenyl can be substituted with one or more of halogen,C₁-C₈ alkyl, NO₂, CF₃, OCF₃, OCF₂H, CN, (CR⁵R⁶)_(c)N(R⁷)₂, wherein c is0 or an integer from 1 to 5.

In another aspect of this embodiment, R is amino or

In yet another aspect of this embodiment, R¹⁷ is isopropyl; in oneembodiment, R¹⁸ is methyl.

Exemplary compounds that are persistent sodium channel antagonistsuseful in a method of the invention are shown as Formulas 1, 2, 3 and 4.In addition, the compounds shown in FIG. 1 have selectivities forpersistent sodium current of 32-fold, 38-fold, 110-fold, and 453-fold,relative to transient sodium current.

As used herein, the term “alkyl” means a straight-chain, branched orcyclic saturated aliphatic hydrocarbon. For example, an alkyl group canhave 1 to 12 carbons, such as from 1 to 7 carbons, or from 1 to 4carbons. Exemplary alkyl groups include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.An alkyl group may be optionally substituted with one or moresubstituents are selected from the group consisting of hydroxyl, cyano,alkoxy, ═O, ═S, NO₂, halogen, dimethyl amino, and SH.

As used herein, the term “alkenyl” means a straight-chain, branched orcyclic unsaturated hydrocarbon group containing at least onecarbon-carbon double bond. For example, an alkenyl group can have 1 to12 carbons, such as from 1 to 7 carbons, or from 1 to 4 carbons. Analkenyl group can optionally be substituted with one or moresubstituents. Exemplary substituents include hydroxyl, cyano, alkoxy,═O, ═S, NO₂, halogen, dimethyl amino, and SH.

As used herein, the term “alkynyl” means a straight-chain, branched orcyclic unsaturated hydrocarbon containing at least one carbon-carbontriple bond. For example, an alkynyl group can have 1 to 12 carbons,such as from 1 to 7 carbons, or from 1 to 4 carbons. An alkynyl groupcan optionally be substituted with one or more substituents. Exemplarysubstituents include hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen,dimethyl amino, and SH.

As used herein, the term “alkoxyl” means an “O-alkyl” group.

As used herein, the term “aryl” means an aromatic group which has atleast one ring having a conjugated pi electron system and includescarbocyclic aryl, heterocyclic aryl and biaryl groups. An aryl group canoptionally be substituted with one or more subtituents. Exemplarysubstituents include halogen, trihalomethyl, hydroxyl, SH, OH, NO₂,amine, thioether, cyano, alkoxy, alkyl, and amino.

As used herein, the term “alkaryl” means an alkyl that is covalentlyjoined to an aryl group. The alkyl can be, for example, a lower alkyl.

As used herein, the term “carbocyclic aryl” means an aryl group whereinthe ring atoms are carbon.

As used herein, the term “heterocyclic aryl” means an aryl group havingfrom 1 to 3 heteroatoms as ring atoms, the remainder of the ring atomsbeing carbon. Heteroatoms include oxygen, sulfur, and nitrogen. Thus,heterocyclic aryl groups include furanyl, thienyl, pyridyl, pyrrolyl,N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like.

As used herein, the term “hydrocarbyl” means a hydrocarbon radicalhaving only carbon and hydrogen atoms. For example, an hydrocarbylradical can have from 1 to 20 carbon atoms, such as from 1 to 12 carbonatoms or from 1 to 7 carbon atoms.

As used herein, the term “substituted hydrocarbyl” means a hydrocarbylradical wherein one or more, but not all, of the hydrogen and/or thecarbon atoms are replaced by a halogen, nitrogen, oxygen, sulfur orphosphorus atom or a radical including a halogen, nitrogen, oxygen,sulfur or phosphorus atom, e.g. fluoro, chloro, cyano, nitro, hydroxyl,phosphate, thiol, etc.

As used herein, the term “amide” means —C(O)—NH—R′, wherein R′ is alkyl,aryl, alkylaryl or hydrogen. As used herein, the term “thioamide” means—C(S)—NH—R′, wherein R′ is alkyl, aryl, alkylaryl or hydrogen. As usedherein, the term “amine” means a —N(R″)R′″ group, wherein R″ and R′″ areindependently selected from the group consisting of alkyl, aryl, andalkylaryl. As used herein, the term “thioether” means —S—R″, wherein R″is alkyl, aryl, or alkylaryl. As used herein, the term “sulfonyl” refersto —S(O)₂—R″″, where R″″ is aryl, C(CN)═C-aryl, CH₂CN, alkyaryl,sulfonamide, NH-alkyl, NH-alkylaryl, or NH-aryl.

IV. Screening Assays

The ability of a compound to selectively reduce persistent sodiumcurrent relative to transient sodium current can be determined using avariety of assays. Such assays can be performed, for example, in a cellor tissue that expresses an endogenous or recombinantly expressedpersistent sodium current, and generally involve determining persistentand transient sodium current prior to and following application of atest compound.

Methods for measuring sodium current are well known to those skilled inthe art, and are described, see, e.g., Joseph S. Adorante, Inhibition ofNoninactivating Na Channels of Mammalian Optic Nerve as a Means ofPreventing Optic Nerve Degeneration Associated with Glaucoma, U.S. Pat.No. 5,922,746 (Jul. 13, 1999); Bert Sakmann & Erwin Neher, SINGLECHANNEL RECORDING (Plenum Press, 2^(nd) ed. 1995); and Tsung-Ming Shihet al., High-level Expression and Detection of Ion Channels in XenopusOocytes, 529-556 (METHODS IN ENZYMOLOGY: ION CHANNELS PART B, Vol. 293,P. Michael Conn ed., Academic Press 1998), which are hereby incorporatedby reference in their entirety. These protocols are routine procedureswell within the scope of one skilled in the art and from the teachingherein (see, e.g., Examples 1, 2, 3 and 4). Since the rate at whichsodium currents open and close is rapid and the speed at which ions flowthrough the channel is high, channel function can be studied using anelectrophysiological approach, which is capable of measuring the ionflux at the rate of one million ions per second with a millisecond timeresolution. In addition, as shown in Examples 1, 2 and 3, a method foridentifying a selective persistent sodium channel antagonist or otherpersistent sodium current antagonist can involve using a fluorescent dyethat is sensitive to change in cell membrane potential in order toenable optical measurement of cell membrane potential. As disclosedherein below, a compound to be tested is added to a well containingcells that express a sodium channel capable of mediating a persistentsodium current, and express a potassium channel or a sodium/potassiumATPase or both.

Methods for measuring membrane potential with voltage-sensitive dyes arewell known to those skilled in the art, and are described, see, e.g.,lain D. Johnson, Fluorescent Probes for Living Cells 30(3) HISTOCHEM. J.123-140 (1998); and I_(MAGING) NEURONS: A LABORATORY MANUAL (RafaelYuste, et al., eds., Cold Spring Harbor Laboratory Press, 2000). Inparticular, the example listed below takes advantage of the hightemporal and spatial resolution that derives from utilization offluorescence resonance energy transfer (FRET) in the measurement ofmembrane potential by voltage-sensitive dyes as described, see, e.g.,Jesus E. Gonzalez & Roger Y. Tsien, Improved Indicators of Cell MembranePotential That Use Fluorescence Resonance Energy Transfer 4(4) CHEM.BIOL. 269-277 (1997); Roger Y. Tsien & Jesus E. Gonzalez, VoltageSensing by Fluorescence Resonance Energy Transfer, U.S. Pat. No.5,661,035 (Aug. 26, 1997); Roger Y. Tsien & Jesus E. Gonzalez, Detectionof Transmembrane Potentials by Optical Methods, U.S. Pat. No. 6,342,379(Jan. 29, 2002); Jesus E. Gonzalez & Michael P. Maher, CellularFluorescent Indicators and Voltage/Ion Probe Reader (VIPR) Tools for IonChannel and Receptor Drug Discovery, 8(5-6) RECEPTORS CHANNELS 283-295,(2002); and Michael P. Maher & Jesus E. Gonzalez, High Throughput Methodand System for Screening Candidate Compounds for Activity Against TargetIon Channels, U.S. Pat. No. 6,686,193 (Feb. 3, 2004), which are herebyincorporated by reference in their entirety.

In addition, the selectivity of a compound for persistent sodium currentversus transient sodium current can be confirmed, as shown in theteaching herein (see, e.g., Examples 2 and 3).

A variety of cell types, including naturally occurring cells andgenetically engineered cells can be used in an in vitro assay to detectpersistent sodium current. Naturally occurring cells havingnon-inactivating sodium current include, for example, several types ofneurons, such as squid axon, cerebellar Purkinje cells, neocorticalpyramidal cells, thalamic neurons, CA1 hipppocampal pyramidal cells,striatal neurons and mammalian CNS axons. Other naturally occurringcells having persistent sodium current can be identified by thoseskilled in the art using methods disclosed herein below and other wellknown methods. Cells for use in testing a compound for its ability toalter persistent sodium current can be obtained from a mammal, such as amouse, rat, pig, goat, monkey or human, or a non-mammal containing acell expressing a sodium channel capable of mediating persistent sodiumcurrent.

Genetically engineered cells having persistent sodium current cancontain, for example, a cDNA encoding a sodium channel capable ofmediating a persistent current such as Nav_(v)1.3; or can be a cellengineered to have increased expression of a sodium channel capable ofmediating a persistent current, decreased expression of a sodium channelmediating a transient current, or both. Recombinant expression isadvantageous in providing a higher level of expression of a sodiumchannel capable of mediating a persistent sodium current than is foundendogenously and also allows expression in cells or extracts in whichthe channel is not normally found. One or more recombinant nucleic acidexpression constructs generally contain a constitutive or induciblepromoter of RNA transcription appropriate for the host cell ortranscription-translation system, operatively linked to a nucleotidesequence that encodes one or more polypeptides of the channel ofinterest. The expression construct can be DNA or RNA, and optionally canbe contained in a vector, such as a plasmid or viral vector. Based onwell-known and publicly available knowledge of nucleic acid sequencesencoding subunits of many sodium channels, including several sodiumchannels capable of mediating a persistent sodium current, one skilledin the art can express desired levels of a biologically activepersistent or transient sodium channels using routine laboratory methodsas described, see, e.g., Molecular Cloning A Laboratory Manual (JosephSambrook & David W. Russell eds., Cold Spring Harbor Laboratory Press,3^(rd) ed. 2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FrederickM. Ausubel et al., eds., John Wiley & Sons, 2004), which are herebyincorporated by reference in their entirety. cDNAs for several familiesof sodium channels have been cloned and sequenced, and are described,see, e.g, Alan L. Goldin, Diversity of Mammalian Voltage-gated SodiumChannels, 868 A_(NN.) N.Y. A_(CAD.) SCI. 38-50 (1999), William A.Catterall, From Ionic Currents to Molecular Mechanisms: The Structureand Function of Voltage-gated Sodium Channels, 26(1) NEURON 13-25(2000); John N. Wood & Mark D. Baker, Voltage-gated Sodium Channels,1(1) CURR. OPIN. PHARMACOL. 17-21 (2001); and Frank H. Yu & William A.Catterall, Overview of the Voltage-Gated Sodium Channel Family, 4(3)GENOME BIOL. 207 (2003), which are hereby incorporated by reference intheir entirety. In addition, both nucleotide and protein sequences allcurrently described sodium channels are publicly available from theGenBank database (National Institutes of Health, National Library ofMedicine, http://www.ncbi.nlm.nih.gov/), which is hereby incorporated byreference in its entirety.

Exemplary host cells that can be used to express recombinant sodiumchannels include isolated mammalian primary cells; established mammaliancell lines, such as COS, CHO, HeLa, NIH3T3, HEK 293-T and PC12;amphibian cells, such as Xenopus embryos and oocytes; and othervertebrate cells. Exemplary host cells also include insect cells such asDrosophila, yeast cells such as S. cerevisiae, S. pombe, or Pichiapastoris and prokaryotic cells (such as E. coli,) engineered torecombinantly express sodium channels.

V. Reaction Schemes

A compound used in a method of the invention can be synthesized bygeneral synthetic methodology, such as by the specific syntheticreaction schemes and methodologies described below and in Examples 5, 6,7 and 8. Modifications of these synthetic methodologies will becomereadily apparent to the practicing synthetic organic chemist in view ofthe following disclosure and general knowledge available in the art.

The reaction schemes disclosed below are directed to the synthesis ofexemplary compounds used in a method of the invention. The syntheticprocesses described herein are adaptable within the skill of thepracticing organic chemist and can be used with such adaptation for thesynthesis of compounds useful in a method of the invention that are notspecifically described. Reaction schemes 1, 2, 3 and 4 disclosesynthetic routes to compounds having Formulas 1, 2, 3 and 4,respectively. Examples 5, 6, 7 and 8 describe methodology useful forsynthesizing exemplary compounds representative of Formulas 1, 2, 3 and4, respectively.

The specific reaction conditions described in Examples 5, 6, 7 and 8 aredirected to the synthesis of exemplary compounds useful in a method ofthe invention. Whereas each of the specific and exemplary syntheticmethods shown in Examples 5, 6, 7 and 8 describe specific compoundswithin the scope of general Formulas 1 through 4, the syntheticprocesses and methods used therein are adaptable within the skill of thepracticing organic chemist and can be used with such adaptation for thesynthesis of compounds useful in a method of the invention that are notspecifically described herein as examples.

VI. Pain Models

The ability of a compound that selectively reduces persistent sodiumcurrent relative to transient sodium current to treat chronic pain in amammal can be confirmed using a variety of well-known assays. Suchessays include, but are not limited to, the Mouse Writhing Assay, theTail Flick Assay, the Sciatic Nerve Ligation assay, the Formalin Testand the Dorsal Root Ganglia Ligation assay.

An accepted standard for detecting and comparing the analgesic activityof different classes of analgesic compounds for which there is acorrelation with human analgesic activity is the prevention of aceticacid induced writhing in mice, see, e.g., R. Koster et al., Acetic Acidfor Analgesic Screening, 18 FED. PROC. 412-416 (1959). In the MouseWrithing Assay, mice are treated with various doses of a test compoundor vehicle, followed by intraperitoneal injection with a standardchallenge dose of acetic acid 5 minutes prior to a designatedobservation period. The acetic acid can be prepared as a 0.55% solutionand injected at a volume of 0.1 ml/10 grams of body weight. For scoringpurposes a “writhe” is indicated by whole body stretching or contractingof the abdomen during an observation period beginning about five minutesafter the administration of acetic acid.

Another model that has been used to define or monitor analgesic levelsfollowing exposure to a variety of compounds is the Tail Flick Assay,see, e.g., William L. Dewey et al., The Effect of Narcotics and NarcoticAntagonists on the Tail-Flick Response in Spinal Mice, 21(8) J. PHARM.PHARMACOL. 548-550 (1969). In this assay, an apparatus can be used totest mice, rats or monkeys by focusing a beam of light on the tail andevaluating latency to tail-flick. This test has proven useful forscreening weak and strong analgesics. In the Tail flick Assay, mice aretreated with various doses of a test compound or vehicle. At a selectedtime point after administration, mice are placed in a holding tube andthe time required for each mouse to react (tail flick) to the heat froma beam of light focused on the tail is recorded on a Tail FlickApparatus (Columbus Instruments, Columbus, Ohio).

An accepted model for assessment of neuropathic pain analgesia is theChung model of peripheral neuropathic pain, see, e.g., Sun H. Kim & JinM. Chung, An Experimental Model for Peripheral Neuropathy Produced bySegmental Spinal Nerve Ligation in the Rat, 50(3) PAIN 355-363 (1992),which is hereby incorporated by reference in its entirety. The Chungmodel is a selective spinal neurectomy model that involves introducingpartial nerve injury by performing a spinal nerve ligation procedure.These protocols for this procedure are routine and well within the scopeof one skilled in the art and from the teaching herein (see, e.g.,Example 4).

Another accepted model for assessment of neuropathic pain analgesia isthe Sciatic Nerve Ligation model, see, e.g., Gary J. Bennett and Yi-KuanXie, A Peripheral Mononeuropathy in Rat That Produces Disorders of PainSensation Like Those Seen in Man, 33(1) PAIN 87-107 (1988); and Youn-WooLee et al., Systemic and Supraspinal, but not Spinal, Opiates SuppressAllodynia in a Rat Neuropathic Pain Model, 199(2) NEUROSCI. LETT.186:111-114 (1995), which are hereby incorporated by reference in theirentirety. In the Sciatic Nerve Ligation model, rats are anesthetized anda nerve ligation procedure performed. The common sciatic nerve isexposed and 4 ligatures are tied loosely around it with about 1 mmspacing. One day to 10 weeks after surgery, nociceptive testing isperformed. Responses to noxious heat are determined by placing the ratsin a chamber with a clear glass floor and aiming at the plantar surfaceof the affected foot a radiant heat source from beneath the floor.Increased latency to withdraw the hindpaw is demonstrative of analgesicactivity. Responses to normally innocuous mechanical stimuli aredetermined by placing the rats in a chamber with a screen floor andstimulating the plantar surface of the hind paw with graduated von Freyhairs which are calibrated by the grams of force required to bend them.Rats with sciatic nerve ligation respond to lower grams of mechanicalstimulation by reflexive withdrawal of the foot than unoperated rats,demonstrating allodynia. An increase in the grams of mechanical forcerequired to produce foot withdrawal is demonstrative of anti-allodynicactivity.

The Formalin Test is a well accepted model of inflammatory pain, see,e.g., Annika B. Malmberg & Tony L. Yaksh, Antinociceptive Actions ofSpinal Nonsteroidal Anti-Inflammatory Agents on the Formalin Test in theRat, 263(1) J. PHARMACOL. EXP. THER. 136-146 (1992). Rats areanesthetized, and, following a loss of spontaneous movement, they areinjected subcutaneously in the dorsal surface of the hindpaw with 50microliters of 5% formalin solution using a 30 gauge needle. Rats arethen individually placed in an open Plexiglas chamber for observation,and within a maximum interval of 1 to 2 minutes, the animals displayrecovery from anesthesia with spontaneous activity and normal motorfunction. Pain behavior is quantified by periodically counting theincidents of spontaneous flinching/shaking of the injected paw. Theflinches are counted for 1-minute periods at 1- to 2-, 5- to 6- and 5minute intervals during the interval from 10 to 60 minutes. Inhibitionof the flinching/shaking of the injected paw is demonstrative of ananalgesic activity.

Using any of these assays, those skilled in the art recognize that ED₅₀values and their standard errors of the mean can be determined usingaccepted numerical methods, see, e.g., Roger E. Kirk, EXPERIMENTALDESIGN: PROCEDURES FOR THE B EHAVIORAL SCIENCES, (Wadsworth Publishing,3^(rd) ed. 1994), which is hereby incorporated by reference in itsentirety. One skilled in the art understands that any of the above orother well known models of pain can be useful for corroborating that aselective persistent sodium current antagonist, including a selectivepersistent sodium channel antagonist, is useful for treating chronicpain.

VII. Pharmaceutical Compositions

As disclosed herein, a selective persistent sodium current antagonist isadministered to a mammal to treat chronic pain. As used herein, the term“treating chronic pain,” when used in reference to administering to amammal an effective amount of a selective persistent sodium currentantagonist, means reducing a symptom of chronic pain, or delaying orpreventing onset of a symptom of chronic pain in the mammal. Forexample, the term “treating chronic pain” can mean reducing a symptom ofchronic pain by at least 30%, 40%, 60%, 70%, 80%, 90% or 100%. Theeffectiveness of a selective persistent sodium current antagonist intreating chronic pain can be determined by observing one or moreclinical symptoms or physiological indicators associated with pain. Forexample, a reduction in chronic pain can include an arrest or a decreasein clinical symptoms of chronic pain or physiological indicatorsassociated with chronic pain. A reduction in chronic pain also can beindicated by a reduced need for a concurrent therapy for chronic pain,such as reduced need for analgesic therapy, TENS, counterirritation,trigger point injection, spray and stretch, or physical therapy. Thoseof skill in the art will know the appropriate symptoms or indicatorsassociated with specific types of chronic pain and will know how todetermine if an individual is a candidate for treatment with a selectivepersistent sodium current antagonist.

The appropriate effective amount to be administered for a particularapplication of the methods can be determined by those skilled in theart, using the guidance provided herein. For example, an effectiveamount can be extrapolated from in vitro and in vivo assays as describedherein above. One skilled in the art will recognize that the conditionof the patient can be monitored throughout the course of therapy andthat the effective amount of a selective persistent sodium currentantagonist that is administered can be adjusted accordingly.

The invention also can be practiced by administering an effective amountof persistent sodium current antagonist together with one or more otheragents including, but not limited to, one or more analgesic agents. Insuch “combination” therapy, it is understood that the antagonist can bedelivered independently or simultaneously, in the same or differentpharmaceutical compositions, and by the same or different routes ofadministration as the one or more other agents.

Exemplary compounds that have at least 20-fold selectivity for reducingpersistent sodium current relative to non-persistent sodium currentinclude those shown in Formulas 1, 2, 3 and 4. Also encompassed by theinvention are pharmaceutically acceptable salts, esters and amidesderived from Formulas 1, 2, 3 or 4. Suitable pharmaceutically acceptablesalts of the antagonists useful in the invention include, withoutlimitation, acid addition salts, which can be formed, for example, bymixing a solution of the antagonist with a solution of an appropriateacid such as hydrochloric acid, sulfuric acid, fumaric acid, maleicacid, succinic acid, acetic acid, benzoic acid, citric acid, tartaricacid, carbonic acid or phosphoric acid. Where an antagonist carries anacidic moiety, suitable pharmaceutically acceptable salts thereof caninclude alkali salts such as sodium or potassium salts; alkaline earthsalts such as calcium or magnesium salts; and salts formed with suitableorganic ligands, for example, quaternary ammonium salts. Representativepharmaceutically acceptable salts include, yet are not limited to,acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate,borate, bromide, calcium edetate, camsylate, carbonate, chloride,clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate,esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate,pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate,tosylate, triethiodide and valerate.

Thus, it is understood that the functional groups of antagonists usefulin the invention can be modified to enhance the pharmacological utilityof the compound. Such modifications are well within the knowledge of theskilled chemist and include, without limitation, esters, amides, ethers,N-oxides, and pro-drugs of the indicated antagonist. Examples ofmodifications that can enhance the activity of an antagonist include,for example, esterification such as the formation of C1 to C6 alkylesters, such as C1 to C4 alkyl esters, wherein the alkyl group is astraight or branched chain. Other acceptable esters include, forexample, C5 to C7 cycloalkyl esters and arylalkyl esters such as benzylesters. Such esters can be prepared from the compounds described hereinusing conventional methods well known in the art of organic chemistry.

Other pharmaceutically acceptable modifications include the formation ofamides. Useful amide modifications include, for example, those derivedfrom ammonia; primary C1 to C6 dialkyl amines, where the alkyl groupsare straight or branched chain; and arylamines having varioussubstitutions. In the case of secondary amines, the amine also can be inthe form of a 5- or 6-member ring. Methods for preparing these and otheramides are well known in the art.

It is understood that, where an antagonist useful in the invention hasat least one chiral center, the antagonist can exist as chemicallydistinct enantiomers. In addition, where an antagonist has two or morechiral centers, the compound exists as diastereomers. All such isomersand mixtures thereof are encompassed within the scope of the indicatedantagonist. Similarly, where an antagonist possesses a structuralarrangement that permits the structure to exist as tautomers, suchtautomers are encompassed within the scope of the indicated antagonist.Furthermore, in crystalline form, an antagonist can exist as polymorphs;in the presence of a solvent, an antagonist can form a solvate, forexample, with water or a common organic solvent. Such polymorphs,hydrates and other solvates also are encompassed within the scope of theindicated antagonist as defined herein.

A selective persistent sodium current antagonist or other compounduseful in the invention generally is administered in a pharmaceuticalacceptable composition. As used herein, the term “pharmaceuticallyacceptable” refer to any molecular entity or composition that does notproduce an adverse, allergic or other untoward or unwanted reaction whenadministered to a human or other mammal. As used herein, the term“pharmaceutically acceptable composition” refers to a therapeuticallyeffective concentration of an active ingredient. A pharmaceuticalcomposition may be administered to a patient alone, or in combinationwith other supplementary active ingredients, agents, drugs or hormones.The pharmaceutical compositions may be manufactured using any of avariety of processes, including, without limitation, conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, and lyophilizing. The pharmaceuticalcomposition can take any of a variety of forms including, withoutlimitation, a sterile solution, suspension, emulsion, lyophilizate,tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosageform suitable for administration.

It is also envisioned that a pharmaceutical composition disclosed in thepresent specification can optionally include a pharmaceuticallyacceptable carriers that facilitate processing of an active ingredientinto pharmaceutically acceptable compositions. As used herein, the term“pharmacologically acceptable carrier” refers to any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, auxiliary or excipient.” Such a carriergenerally is mixed with an active compound, or permitted to dilute orenclose the active compound and can be a solid, semi-solid, or liquidagent. It is understood that the active ingredients can be soluble orcan be delivered as a suspension in the desired carrier or diluent. Anyof a variety of pharmaceutically acceptable carriers can be usedincluding, without limitation, aqueous media such as, e.g., distilled,deionized water, saline; solvents; dispersion media; coatings;antibacterial and antifungal agents; isotonic and absorption delayingagents; or any other inactive ingredient. Selection of apharmacologically acceptable carrier can depend on the mode ofadministration. Except insofar as any pharmacologically acceptablecarrier is incompatible with the active ingredient, its use inpharmaceutically acceptable compositions is contemplated. Non-limitingexamples of specific uses of such pharmaceutical carriers can be foundin PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS (Howard C.Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7^(th) ed.1999); REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (Alfonso R.Gennaro ed., Lippincott, Williams & Wilkins, 20^(th) ed. 2000); GOODMAN& GILMAN's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (Joel G. Hardman etal., eds., McGraw-Hill Professional, 10^(th) ed. 2001); and HANDBOOK OFPHARMACEUTICAL EXCIPIENTS (Raymond C. Rowe et al., APhA Publications,4^(th) edition 2003) which are hereby incorporated by reference in theirentirety. These protocols are routine procedures and any modificationsare well within the scope of one skilled in the art and from theteaching herein.

It is further envisioned that a pharmaceutical composition disclosed inthe present specification can optionally include, without limitation,other pharmaceutically acceptable components, including, withoutlimitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed in thepresent specification, provided that the resulting preparation ispharmaceutically acceptable. Such buffers include, without limitation,acetate buffers, citrate buffers, phosphate buffers, neutral bufferedsaline, phosphate buffered saline and borate buffers. It is understoodthat acids or bases can be used to adjust the pH of a composition asneeded. Pharmaceutically acceptable antioxidants include, withoutlimitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. Usefulpreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate and a stabilized oxy chloro composition, for example, PURITE®.Tonicity adjustors useful in a pharmaceutical composition include,without limitation, salts such as, e.g., sodium chloride, potassiumchloride, mannitol or glycerin and other pharmaceutically acceptabletonicity adjustor. The pharmaceutical composition may be provided as asalt and can be formed with many acids, including but not limited to,hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. It is understood that these andother substances known in the art of pharmacology can be included in apharmaceutical composition useful in the invention.

An antagonist useful in a method of the invention is administered to amammal in an effective amount. Such an effective amount generally is theminimum dose necessary to achieve the desired therapeutic effect, whichcan be, for example, that amount roughly necessary to reduce thediscomfort caused by the pain to tolerable levels or to achieve asignificant reduction in pain. For example, the term “effective amount”when used with respect to treating chronic pain can be a dose sufficientto reduce pain, for example, by at least 30%, 40%, 50%, 60%, 70%, 80%,90% or 100%. Such a dose generally is in the range of 0.1-1000 mg/dayand can be, for example, in the range of 0.1-500 mg/day, 0.5-500 mg/day,0.5-100 mg/day, 0.5-50 mg/day, 0.5-20 mg/day, 0.5-10 mg/day or 0.5-5mg/day, with the actual amount to be administered determined by aphysician taking into account the relevant circumstances including theseverity of the chronic pain, the age and weight of the patient, thepatient's general physical condition, the cause of chronic pain and theroute of administration. Where repeated administration is used, thefrequency of administration depends, in part, on the half-life of theantagonist. Suppositories and extended release formulations can beuseful in the invention and include, for example, dermal patches,formulations for deposit on or under the skin and formulations forintramuscular injection. It is understood that slow-release formulationsalso can be useful in the methods of the invention. The subjectreceiving the selective persistent sodium channel antagonist can be anymammal or other vertebrate capable of experiencing chronic pain, forexample, a human, primate, horse, cow, dog, cat or bird.

Various routes of administration can be useful for treating chronic painaccording to a method of the invention. A pharmaceutical compositionuseful in the methods of the invention can be administered to a mammalby any of a variety of means depending, for example, on the type andlocation of chronic pain to be treated, the antagonist or other compoundto be included in the composition, and the history, risk factors andsymptoms of the subject. Routes of administration suitable for themethods of the invention include both systemic and local administration.As non-limiting examples, a pharmaceutical composition useful fortreating chronic pain can be administered orally or by subcutaneouspump; by dermal patch; by intravenous, subcutaneous or intramuscularinjection; by topical drops, creams, gels or ointments; as an implantedor injected extended release formulation; as a bioerodable ornon-bioerodable delivery system; by subcutaneous minipump or otherimplanted device; by intrathecal pump or injection; or by epiduralinjection. An exemplary list of biodegradable polymers and methods ofuse are described in, e.g., Heller, Biodegradable Polymers in ControlledDrug Delivery (CRC CRITICAL REVIEWS IN THERAPEUTIC DRUG CARRIER SYSTEMS,Vol. 1. CRC Press, 1987); Vernon G. Wong, Method for Reducing orPreventing Transplant Rejection in the Eye and Intraocular Implants forUse Therefor, U.S. Pat. No. 6,699,493 (Mar. 2, 2004); Vernon G. Wong &Mae W. L. Hu, Methods for Treating Inflammation-mediated Conditions ofthe Eye, U.S. Pat. No. 6,726,918 (Apr. 27, 2004); David A. Weber et al.,Methods and Apparatus for Delivery of Ocular Implants, U.S. PatentPublication No. US2004/0054374 (Mar. 18, 2004); Thierry Nivaggioli etal., Biodegradable Ocular Implant, U.S. Patent Publication No.US2004/0137059 (Jul. 15, 2004), which are hereby incorporated byreference in their entirety. It is understood that the frequency andduration of dosing will be dependent, in part, on the relief desired andthe half-life of the selective persistent sodium current antagonist.

In particular embodiments, a method of the invention is practiced byperipheral administration of a selective persistent sodium currentantagonist. As used herein, the term “peripheral administration” or“administered peripherally” means introducing an agent into a subjectoutside of the central nervous system. Peripheral administrationencompasses any route of administration other than direct administrationto the spine or brain. As such, it is clear that intrathecal andepidural administration as well as cranial injection or implantation arenot within the scope of the term “peripheral administration” or“administered peripherally.” It further is clear that some selectivepersistent sodium current antagonists can cross the blood-brain barrierand, thus, become distributed throughout the central and peripheralnervous systems following peripheral administration.

Peripheral administration can be local or systemic. Local administrationresults in significantly more of a pharmaceutical composition beingdelivered to and about the site of local administration than to regionsdistal to the site of administration. Systemic administration results indelivery of a pharmaceutical composition to essentially the entireperipheral nervous system of the subject and may also result in deliveryto the central nervous system depending on the properties of thecomposition.

Routes of peripheral administration useful in the methods of theinvention encompass, without limitation, oral administration, topicaladministration, intravenous or other injection, and implanted minipumpsor other extended release devices or formulations. A pharmaceuticalcomposition useful in the invention can be peripherally administered,for example, orally in any acceptable form such as in a tablet, liquid,capsule, powder, or the like; by intravenous, intraperitoneal,intramuscular, subcutaneous or parenteral injection; by transdermaldiffusion or electrophoresis; topically in any acceptable form such asin drops, creams, gels or ointments; and by minipump or other implantedextended release device or formulation.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLES Example 1 High-Throughput Screening Assay for Identification ofInhibitors of Persistent Sodium Current

To identify compounds that inhibit persistent sodium current, a primaryhigh-throughput screen was employed, see, e.g., Joseph S. Adorante etal., High-throughput Screen for Identifying Channel Blockers thatSelectively Distinguish Transient from Persistent Sodium Channels, U.S.Patent Publication No. 2002/0077297 (Jun. 20, 2002), which is herebyincorporated by reference in its entirety.

I. Compound Identification Assay Overview

To examine the ability of test compounds to alter persistent sodiumcurrent, human embryonic kidney (HEK) cells were transfected withNa_(v)1.3 sodium channel to obtain cells that express sodium currentcapable of mediating persistent sodium current. HEK cells expressingNa_(v)1.3 (HEK-Na_(v)1.3) were added to assay plate wells containing aNa⁺-free media and physiologic concentrations of K⁺ (4.5 mM) andpreincubated for 20 minutes with ion-sensitive FRET dyes and either 5 μMof a test compound or a DMSO control. The assay plates were thentransferred to a voltage/ion probe reader (VIPR) (Aurora Biosciences,San Diego, Calif.) and the VIPR was adjusted so that the fluorescentemission ratio from the donor ands acceptor FRET dyes equaled 1.0. Toelicit persistent sodium current, a double addition protocol wasperformed by first adding an isotonic solution to adjust theconcentration of sodium and potassium ions in the well to 110 mM and 10mM, respectively, and measuring the resulting sodium-dependentdepolarization and second by adding K⁺ to a final concentration of 80mM, and measuring K⁺-dependent depolarization. Test compounds that blockthe Na⁺ dependent signal, but not the K⁺ dependent signal were selectedfor further analysis. The Na⁺-dependent depolarization resulting fromthe persistent Na⁺ was measured as shown in FIG. 2. The labeled boxesindicate the application of Na+ or K⁺. Circles indicate the controlresponse with 0.1% DMSO added, triangles show the effects of the Na⁺channel inhibitor tetracaine (10 μM), and the diamonds show the responseduring the application of a non-specific channel blocker.

In this high-throughput assay, non-specific agents that inhibit membranedepolarizations induced by any effector must be distinguished from truepersistent Na⁺ current antagonists, which block only Na⁺-dependentdepolarizations. Therefore, a counter-screen to determine the ability ofcompounds to alter K⁺-dependent depolarization was performed. As shownin FIG. 2, following pre-incubation with vehicle alone (DMSO) both Na⁺and K⁺ additions produced a robust depolarization as indicated by theincrease in Rf/Ri. Tetracaine, a Na⁺ channel blocker, inhibited theNa⁺-dependent, but not the K⁺-dependent change in Rf/Ri. In contrast, anon-specific inhibitor of Na⁺ and K⁺-dependent depolarization blockedthe change in Rf/Ri following either addition. This data demonstratesthat selective antagonists of the persistent sodium current can beidentified using the described method.

To eliminate compounds that non-specifically inhibited the Na⁺-dependentdepolarization, data obtained using the above procedure was analyzedwith respect to a counter-screen that used K⁺-dependent depolarizationas a readout. To select hits from the primary screen, the data wereplotted as histograms. Inhibition of the Na⁺-dependent depolarizationwas plotted against inhibition of the K⁺-dependent depolarization. Basedon these data, the criteria for selection as a hit, was a greater orequal to 90% inhibition of the Na⁺-dependent depolarization and a lessthan or equal to 20% inhibition of the K⁺-dependent depolarization. Thisprotocol provided a distinction between compounds that were inert ornon-specific in their effects and compounds that specifically block thepersistent sodium current.

II. Solutions

Solution compositions and volumes used in the assay are described below.Functions of some components of the solutions using the assay are asfollows: (1) CC2-DMPE: a stationary coumarin-tagged phospholipidresonance energy donor. This dye is excited at 405 nm wavelength lightand in the absence FRET emits fluorescence at 460 nm. (2) DiSBAC2 (3) orDiSBAC6(3): mobile resonance energy acceptors that partition across themembrane as a function of the electric field. The excitation spectra forthese dyes overlap the emission of the coumarin donor and, thus, theyact as FRET acceptors. They have an emission spectrum in the range of570 nm. (3) ESS-AY17: reduces the background fluorescence thatcomplicates the assay. (4) CdCl₂ (400 μM) was included in thepre-incubation solutions to stabilize the membrane potential of thecells at negative resting potential, resulting in the maximum number ofNa⁺ channels being available for activation. (5) Extracellular Cl— wasreplaced with MeSO₃ during preincubation and throughout the assay. Thiseliminates a complicating Cl— current during the assay and results in anamplified and more stable voltage-change induced by the persistent Na⁺current. (6) 1st K⁺ addition: functions to depolarize the test cells toa voltage that activates substantial numbers of Na⁺ channels. (7) 2nd K⁺addition: this addition produces a K⁺-dependent depolarization, which isused as a counterscreen to eliminated non-specific blockers.

III. Cell Culture

HEK-293 cells were grown in Minimum Essential Medium (Invitrogen, Inc.,Carlsbad, Calif.) supplemented with 10% Fetal Bovine Serum (Invitrogen,Inc., Carlsbad, Calif.) and 1% Pennicillin-Streptomycin (Invitrogen,Inc., Carlsbad, Calif.). Medium for HEK-Na_(v)1.3 cells also contained500 mg/ml G418 Geneticin (Invitrogen, Inc., Carlsbad, Calif.) and 2 μMTTX (Calbiochem, Inc., San Diego, Calif.) for maintaining selectivepressure. Cells were grown in vented cap flasks, in 90% humidity and 10%CO₂, to about 80% confluence and generally split by trypsinization 1:5or 1:10.

HEK-Na_(v)1.3 cells were seeded in 96-well plates (Becton-Dickinson, SanDiego, Calif.) coated with Matrigel (Becton-Dickinson, San Diego,Calif.) at 40,000 cells (in 100 μl culture medium) per well, and assayedthe following day (16-20 hours). Cells were sometimes incubated in96-well plates at somewhat lower densities (20,000 per well), andincubated for up to 40-48 hours.

IV. HEK-Na_(v)1.3 Handling and Dye Loading

Approximately 16 to 24 hours before the assay, HEK-Na_(v)1.3 cells wereseeded in 96-well poly-lysine coated plates at 40,000 per well. On theday of the assay, medium was aspirated and cells were washed 3 timeswith 150 uL of Bath Solution #1 (BS#1) using CellWash (ThermoLabSystems, Franklin, Mass.).

A 20 μM CC2-DMPE solution was prepared by mixing coumarin stock solutionwith 10% Pluronic 127 1:1 and then dissolving the mix in the appropriatevolume of BS#1. After the last wash, 50 ml of 20 μM CC2-DMPE solutionwas added to 50 mL of residual bath in each well to make 10 μM coumarinstaining buffer. Plates were incubated in the dark for 30-60 minutes atroom temperature.

While the cells were being stained with coumarin, a 10 μM DiSBAC2(3)solution in TEA-MeSO3 bath was prepared. In addition to oxonol, thissolution contained any drug(s) being tested, at 4 times the desiredfinal concentration (e.g. 20 μM for 5 μM final), 1.0 mM ESS-AY17, and400 μM CdCl₂.

After 30-60 minutes of CC2-DMPE staining, the cells were washed 3 timeswith 150 μL of TEA-MeSO₃ buffer. Upon removing the bath, the cells wereloaded with 80 μL of the DiSBAC2(3) solution and incubated for 20-30minutes as before. Typically, wells in one column on each plate (e.g.column 11) were free of test drug(s) and served as positive and negativecontrols.

Once the incubation was complete, the cells were ready to be assayed onVIPR for sodium addback. 240 μL of NaMeSO3 buffer was added to stimulatethe cells, resulting in a 1:4 dilution of the drugs; 240 μL of TEA-MeSO₃buffer or 1 μM TTX was used as a positive control.

V. VIPR Instrumentation and Data Process

Optical experiments in microtiter plates were performed on theVoltage/Ion Probe Reader (VIPR) using two 400 nm excitation filters andfilter sticks with 460 nm and 570 nm filters on the emission side forthe blue and red sensitive PMTs, respectively. The instrument was run incolumn acquisition mode with 2 or 5 Hz sampling and 30 seconds ofrecording per column. Starting volumes in each well were 80 ml; usually240 mL was added to each well during the course of the experiment. Thelamp was allowed to warm up for about 20 minutes, and power to the PMTswas turned on for about 10 minutes prior to each experiment.

Ratiometric measurements of changes in fluorescent emissions at 460- and570 nm on the VIPR platform (Aurora Bioscience, San Diego, Calif.)demonstrated that this assay format produces a robust and reproduciblefluorescent signal upon depolarization of HEK-Na_(v)1.3 cells with aNa⁺/K⁺ addition. From a normalized ratio of 1.0 in Na⁺-free media,Na⁺-dependent depolarization resulted in an increase in the 460/570ratio to over 2.2 (FIG. 2). Inter-well analysis of the ratios indicatedthat the amplitude of signal was large enough and consistent enough tobe used in high-throughput screening.

Data were analyzed and reported as normalized ratios of intensitiesmeasured in the 460 nm and 580 nm channels. The VIPR sampling ratevaried between 2 and 5 Hz in different experiments, with 5 Hz used forhigher resolution of the peak sodium responses. The process ofcalculating these ratios was performed as follows. On all plates, column12 contained TEA-MeSO₃ buffer with the same DiSBAC2(3) and ESS-AY17concentrations as used in the cell plates; however no cells wereincluded in column 12. Intensity values at each wavelength were averagedfor the duration of the scan. These average values were subtracted fromintensity values in all assay wells. The initial ratio obtained fromsamples 5-10 (Ri) was defined as:${Ri} = \frac{{Intensity}_{{460\quad{nm}},\quad{{samples}\quad 5\text{-}10}} - {background}_{460\quad{nm}}}{{Intensity}_{{580\quad{nm}},\quad{{samples}\quad 5\text{-}10}} - {background}_{580\quad{nm}}}$

and the ratio obtained from sample f (Rf) was defined as:${Rf} = \frac{{Intensity}_{{460\quad{nm}},\quad{{sample}\quad f}} - {background}_{460\quad{nm}}}{{Intensity}_{{580\quad{nm}},\quad{{sample}\quad f}} - {background}_{580\quad{nm}}}$

Final data were normalized to the starting ratio of each well andreported as Rf/Ri. The fluorescent response in the Na_(v)1.3 persistentcurrent assay reached a peak approximately 10 seconds following thestart of the run, therefore, the maximum ratio was selected as thereadout for the assay (FIG. 3).

VI. Assay Reproducibility and Resolution

The assay format described above allows for quality assurance bymeasuring both negative (DMSO 0.1%) and positive (tetracaine 10 μM)controls. Every 10th plate in an assay run was a control plate. The datafrom these plates were used to verify that the assay conditions wereoptimal and to normalize the data from the test compounds. FIG. 3 showsresults from control plates from multiple assays.

In FIG. 3, control plates having wells containing either 0.1% DMSO or 10μM tetracaine were run after every ninth assay plate. The response toNa⁺-dependent depolarization was measured and the data were binned intohistograms as shown. The mean maximum response (Max) obtained in thepresence of (0.1% DMSO) and the mean minimum response (Min) obtained inthe presence of 10 μM tetracaine were determined. For quality control,data variance was compared to the difference between the maximum andminimum signals. This was accomplished by calculating a screening window(z) for each control plate. Data for the run was accepted if 1.0≧Z≧0.5.$Z = {1 - \frac{{3 \times {STD}_{\max}} + {3 \times {STD}_{\min}}}{{Mean}_{\max} - {Mean}_{\min}}}$

Example 2 Moderate-Throughput Screening Assay for Selectivity ofInhibitors of Persistent Sodium Current

Compounds obtained by the high-throughput screening described in Example1 were tested for selectivity of blockade of persistent sodium currentwith respect to blockade of transient sodium current using amoderate-throughput screen. The selectivity assay utilizes Estimtechnology (Aurora Bioscience, San Diego, Calif.) to induce channelactivation. This assay has an inherently greater time resolution thanthe high-throughput assay, and thus allows the measurement of both thetransient and persistent components of the Na⁺ currents within a singleexperiment.

I. Compound Selectivity Assay Overview

The Estim technology involves instrumenting 96-well plates withelectrodes so that application of an appropriate voltage gradient acrossthe well (electric field stimulation, EFS) can be used for activation ofthe ion channels in the target cells. EFS of HEK-293 cells expressingNa_(v)1.3 channels resulted in a rapid depolarization followed by adelayed repolarization. The transient Na⁺ current drives the rapiddepolarization while the persistent Na⁺ current sustains the delayedrepolarization. When similar experiments were performed in cellsexpressing channels that do not exhibit persistent currents, only rapiddepolarization was seen. For quantification of the block of transientcurrent, the amplitude of peak response was averaged for seven stimuli.The average response was converted to activity by normalizing againstthe difference between the responses in Ringer's solution with DMSO andRinger's solution containing 10 μM tetracaine. Persistent currentactivity was calculated by integrating under the curve. The areaobtained for each compound was normalized against the responses obtainedwith the DMSO control and in the presence of 10 μM tetracaine.

II. Cell Culture

Approximately 16 to 24 hours before the assay, HEK-Na_(v)1.3 cells wereseeded in 96-well poly-lysine coated plates at 60,000 per well. On theday of the assay, medium was aspirated were cells were washed 3 timeswith 150 μL of HBSS using CellWash (Thermo LabSystems, Franklin, Mass.).

III. HEK-Na_(v)1.3 Handling and Dye Loading

A 20 μM CC2-DMPE solution was prepared by mixing coumarin stock solutionwith 10% Pluronic 127 1:1 and then dissolving the mix in the appropriatevolume of HBSS. After the last wash, 50 μL of 20 μM CC2-DMPE solutionwas added to 50 μL of residual bath in each well to make 10 μM coumarinstaining buffer. Plates were incubated in the dark for 30 minutes atroom temperature.

While the cells were being stained with CC2-DMPE, a 0.2 μM DiSBAC6(3)solution in HBSS was prepared.

After 30 minutes of CC2-DMPE staining, the cells were washed 3 timeswith 150 μL of HBSS. After the last wash, 50 μL of 0.2 μM DiSBAC6(3)solution was added to 50 μL of residual bath in each well to make 0.1 μMoxonol staining buffer. Plates were then incubated in the dark for 15minutes.

After 15 minutes of DiSBAC6(3) staining, the cells were washed again 3times with 150 μL of HBSS. After the last wash, 50 μL of 1.0 μM ESS-AY17solution was added to 50 μL of residual bath in each well to make 0.5 μMESS. This solution also contained any drug(s) being tested, at twice thedesired final concentrations. Plates were incubated in the dark againfor 15 minutes. Once the incubation was complete, the cells were assayedon EFS/VSP reader.

III. Fast FRET Reader Instrumentation and Data Process

Optical experiments in microtiter plates were performed on the fast FRETReader using two 400 nm excitation filters and filter sticks with 460 nmand 580 nm filters on the emission side for the blue and red sensitivePMTs, respectively. The instrument was run in column acquisition modewith 100 Hz sampling and 12 seconds of recording per column. Sevenpulses were applied at 1 Hz, starting at 2 seconds. The lamp was allowedto warm up for about 20 minutes, and power to the PMTs was turned on forabout 10 minutes prior to each experiment.

Data were analyzed and reported as normalized ratios of intensitiesmeasured in the 460 nm and 580 nm channels. The process of calculatingthese ratios was performed as follows. On all plates, column 12contained HBSS with the same ESS-AY17 concentration as used in the cellplates; however no cells were included in column 12. Intensity values ateach wavelength were averaged for the duration of the scan. Theseaverage values were subtracted from intensity values in all assay wells.The initial ratio obtained from samples 50-100 (Ri) was defined as:${Ri} = \frac{{Intensity}_{{460\quad{nm}},\quad{{samples}\quad 50\text{-}100}} - {background}_{460\quad{nm}}}{{Intensity}_{{580\quad{nm}},\quad{{samples}\quad 50\text{-}100}} - {background}_{580\quad{nm}}}$

and the ratio obtained from sample f (Rf) was defined as:${Rf} = \frac{{Intensity}_{{460\quad{nm}},\quad{{sample}\quad f}} - {background}_{460\quad{nm}}}{{Intensity}_{{580\quad{nm}},\quad{{sample}\quad f}} - {background}_{580\quad{nm}}}$

Data were normalized to the starting ratio of each well and reported asRf/Ri. The transient Na⁺-current signal was calculated as average of thepeaks resulting from the seven electric pulses applied in the course ofrecording. The persistent Na⁺-current signal was calculated integratingthe area under the total response during the seven electric pulsesapplied in the course of recording. Selectivity was determined bycomparison of concentrations of agent required to block 50% of thepersistent current (IC₅₀) vs. the IC₅₀ for the transient current.

Example 3 Electrophysiological Assay for Selectivity of Inhibitors ofPersistent Sodium Current

To confirm the blocking selectivity of test compounds for persistentsodium current, individual compounds were examined using a whole-cellpatch clamp method.

HEK cells transfected with Na_(v)1.3 sodium channels that expresstransient and persistent sodium currents were plated onto glasscoverslips and cultured in MEM cell culture media with Earle's salts andGlutaMAX (Invitrogen, Inc., Carlsbad, Calif.) supplemented with:10%Fetal bovine serum, heat inactivated (Invitrogen, Inc., Carlsbad,Calif.), 0.1 mM MEM non-essential amino acids (Invitrogen, Inc.,Carlsbad, Calif.), 10 mM HEPES (Invitrogen, Inc., Carlsbad, Calif.), 1%Penicillin/Streptomycin (Invitrogen, Inc., Carlsbad, Calif.).

After an incubation period of from 24 to 48 hours the culture medium wasremoved and replaced with external recording solution (see below). Wholecell patch clamp experiments were performed using an EPC10 amplifier(HEKA Instruments, Lambrecht, Germany.) linked to an IBM compatiblepersonal computer equipped with PULSE software. Borosilicate glass patchpipettes were pulled to a fine tip on a P90 pipette puller (SutterInstrument Co., Novato, Calif.) and were polished (Microforge,Narishige, Japan) to a resistance of about 1.5 Mohm when filled withintracellular recording solution (Table 1). TABLE 1 Patch ClampSolutions External Recording Internal Recording Solution SolutionCompound Concentration Compound Concentration NaCl 127 mM CsMeSO₃ 125 mMHEPES (free acid) 10 mM CsCl 25 mM KCl 5 mM NaHEPES 10 mM CsCl 5 mMAmphotericin 240 μg/ml Glucose 10 mM MgCl₂ 0.6 mM CaCl₂ 1.2 mM CdCl₂ 200μM pH to 7.4 with NaOH @ room temp. pH 7.20 with CsOH300 mOsm 290 mOsm.

Persistent and transient currents in HEK cells expressing Na_(v)1.3channels were measured by applying 200-msec depolarizations from aholding potential of −90 mV to 0 mV. Background currents that remainedin the presence of 500 nM TTX were subtracted from all traces. Drugswere perfused directly into the vicinity of the cells using amicroperfusion system.

Under control conditions, depolarizing pulses elicited a large transientinward current that declined to a smaller persistent current, whichremained stable during the remainder of the pulse (FIG. 4, control).Addition of 500 nM TTX completely blocked both the transient andpersistent currents (FIG. 4, TTX). Application of 3 μM of Compound 1,produced a much different effect. Inspection of FIG. 4 reveals that theCompound 1 blocked 99% of the persistent current while only reducing thetransient current by 16%. Dose-response analysis for Compound 1demonstrates its significant selectivity for blocking the persistentsodium current relative to the transient sodium current over a fourorder of magnitude range (FIG. 5).

Example 4 Administering a Selective Persistent Sodium Current Antagonistin a Rodent Model Results in Reduced Pain

This example describes reversal of allodynia in an animal model ofneuropathic pain by administering a selective persistent sodium channelantagonist.

Compound 1 was tested in a rodent model of neuropathic pain known to bepredictive of clinical activity, see, e.g., Kim & Chung, supra, (1992).Following ligation of two spinal nerves, the animals developedsensitivity to normally non-painful stimuli such as touch. The abilityof Compound 1 to reverse this sensitivity, called allodynia, was tested30 minutes after dosing by intraperitoneal administration. As shown inFIG. 6, Compound 1 produced an 80% reduction in allodynia with respectto a vehicle control.

The animal model used involved the surgical ligation of the L5 (andoptionally the L6) spinal nerves on one side in experimental animals.Rats recovering from the surgery gained weight and displayed a level ofgeneral activity similar to that of normal rats. However, these ratsdeveloped abnormalities of the foot in which the hindpaw was moderatelyeverted and the toes were held together. More importantly, the hindpawon the side affected by the surgery became sensitive to pain fromlow-threshold mechanical stimuli, such as that producing a faintsensation of touch in a human, within about 1 week following surgery.This sensitivity to normally non-painful touch is called “tactileallodynia” and lasts for at least two months. The response includeslifting the affected hindpaw to escape from the stimulus, licking thepaw and holding it in the air for many seconds. None of these responsesis normally seen in the control group.

Rats were anesthetized before surgery. The surgical site was shaved andprepared either with betadine or Novacaine. Incision was made from thethoracic vertebra XIII down toward the sacrum. Muscle tissue wasseparated from the spinal vertebra (left side) at the L4-S2 levels. TheL6 vertebra was located and the transverse process was carefully removedwith a small rongeur to expose the L4-L6 spinal nerves. The L5 and L6spinal nerves were isolated and tightly ligated with 6-0 silk thread.The same procedure was performed on the right side as a control, exceptthat no ligation of the spinal nerves was performed.

A complete hemostasis was confirmed, then the wounds were sutured. Asmall amount of antibiotic ointment was applied to the incised area, andthe rat was transferred to the recovery plastic cage under a regulatedheat-temperature lamp. On the day of the experiment, at least seven daysafter the surgery, six rats per test group were administered the testdrugs by intraperitoneal (i.p.) injection. For i.p. injection, Compound#1 was formulated in approximately 50% DMSO and given in a volume of 1ml/kg body weight. Compound #1 was tested 10 mg/kg.

Tactile allodynia was measured prior to and 30 minutes after drugadministration using von Frey hairs, which are a series of fine hairswith incremental differences in stiffness. Rats were placed in a plasticcage with a wire mesh bottom and allowed to acclimate for approximately30 minutes. The von Frey hairs were applied perpendicularly through themesh to the mid-plantar region of the rats' hindpaw with sufficientforce to cause slight buckling and held for 6-8 seconds. The appliedforce has been calculated to range from 0.41 to 15.1 grams. If the pawis sharply withdrawn, it is considered a positive response. A normalanimal will not respond to stimuli in this range, but a surgicallyligated paw will be withdrawn in response to a 1-2 gram hair.

In summary, results shown in this example indicate that a selectivepersistent sodium current antagonist can be used to effectively reducepain in a mammal.

Example 5 Synthesis of Exemplary Compounds Representative of Formula 1

A compound having general Formula 1, exemplified bythiophene-2-carboxylic acid (4-phenyl-butyl)-amide (Compound 1; FIG. 1)can be prepared as follows. A solution of thiophene-2-carbonyl chloride(147 mg, 1.0 mmol), triethylamine (101 mg, 1.0 mmol) in dichloromethaneis treated with 4-phenylbutylamine (149 mg, 1.0 mmol). The reactionmixture is stirred until no further reaction occurs and is quenched bythe addition of aqueous NaHCO₃ solution. The organic phase is collectedand concentrated to give the title compound.

Example 6 Synthesis of Exemplary Compounds Representative of Formula 2

A compound having general Formula 2, exemplified by1-Benzyl-4-(5-phenyl-[1,3,4]oxadiazol-2-yl)-pyridine (Compound 2;FIG. 1) can be prepared as follows. A solution of4-(5-phenyl-[1,3,4]oxadiazol-2-yl)-pyridine (223 mg, 1.0 mmol) isprepared by the method of H. Smith Broadbent, et al., Quinoxalines. I.Preparation and Stereochemistry of Decahydroquinoxalines, 82(1) J. AMER.CHEM. SOC. 189-193 (1960) in chloroform is treated with benzylbromide(171 mg, 1.0 mmol). The reaction is stirred until no further reactionoccurs. The reaction mixture is concentrated to give the title compound.

Example 7 Synthesis of Exemplary Compounds Representative of Formula 3

A compound having general Formula 3, exemplified by6-Isopropyl-3-methyl-2-{4-[(4-propoxy-benzylidene)-amino]-benzylidene}-cyclohexanone(Compound 3; FIG. 1) can be prepared as follows. A solution of menthone(154 mg, 1.0 mmol) and 4-aminobenzaldehyde (121 mg, 1.0 mmol) indimethylsulfoxide is treated with potassium hydroxide (56 mg, 1.0 mmol).The reaction is stirred until no further reaction occurs. The reactionmixture is poured into ethyl acetate and water. The organic phase iscollected, dried and concentrated to give2-(4-Amino-benzylidene)-6-isopropyl-3-methyl-cyclohexanone. The2-(4-Amino-benzylidene)-6-isopropyl-3-methyl-cyclohexanone is dissolvedin dichloromethane and treated with 4-propoxybenzaldehyde (164 mg, 1.0mmol) and anhydrous Na₂SO₄. The reaction mixture is stirred until nofurther reaction occurs. The reaction mixture is filtered andconcentrated to give the title compound.

Example 8 Synthesis of Exemplary Compounds Representative of Formula 4

A compound having general Formula 4, exemplified by3-(2,2,2-Trifluoro-acetylamino)-benzoic acid 2-oxo-2-phenyl-ethyl ester(Compound 4; FIG. 1) can be prepared as follows. A solution of3-aminobenzoic acid (137 mg, 1.0 mmol) in dichloromethane is treatedwith trifluoroacetic anhydride (420 mg, 2.0 mmol). The reaction mixtureis stirred until no further reaction occurs. The reaction mixture isconcentrated to give 3-(2,2,2-Trifluoro-acetylamino)-benzoic acid. Asolution of 3-(2,2,2-Trifluoro-acetylamino)-benzoic acid (233 mg, 1.0mmol) and 2-hydroxyacetophenone (136 mg, 1.0 mmol) in dimethylformamideand diisopropylethylamine (260 mg, 2.0 mmol) is treated with HBTU (379mg, 1.0 mmol). The reaction mixture is stirred until no further reactionoccurs. The reaction is poured into ethyl acetate and water. The organicphase is collected, dried and concentrated to give the title compound.

Example 9 Oral Administration of a Persistent Sodium Current Blocker toTreat Neuropathic Pain from Trigeminal Neuralgia

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat a neuralgic pain. While the example illustratesthe use of a PSCB to treat trigeminal neuralgia, any acute spasmodicpain that travels along one or more nerves, such as, e.g., post-herpeticneuralgia, glossopharyngeal neuralgia, sciatica and atypical facialpain, can also be treated using this method.

A patient presents pain symptoms that are diagnosed as trigeminalneuralgia. She describes the pain as a sudden sharp stabbing pain on theright side of her face, eyes and lips. The pain is triggered when shetries to chew her food while eating and each episode lasts for severalseconds and may repeat many times over the course of the day. Thatpatient is treated orally with a therapeutically-effective amount of apharmaceutically acceptable composition comprising a PSCB. Within oneday after the administration of a PSCB therapy, the patient's pain issubstantially alleviated. Repeated administration of the PSCBcomposition maintains this pain relief.

Example 10 Oral Administration of a Persistent Sodium Current Blocker toTreat Neuropathic Pain from Phantom Pain

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat a deafferentation pain syndrome. While theexample illustrates the use of a PSCB to treat phantom pain, any painresulting from a loss of the sensory input from a portion of the body,such as, e.g., an injury to the brain, spinal cord, or a peripheralnerve, post-stroke pain, phantom pain, paraplegia, brachial plexusavulsion and postherpetic neuralgia, can also be treated using thismethod.

A patient with an amputated right arm presents symptoms that arediagnosed as phantom pain. That patient is treated orally with atherapeutically-effective amount of a pharmaceutically acceptablecomposition comprising a PSCB. Within one day after the administrationof a PSCB therapy, the patient's pain is substantially alleviated.Repeated administration of the PSCB composition maintains this painrelief.

Example 11 Oral Administration of a Persistent Sodium Current Blocker toTreat Neuropathic Pain from Chemotherapy Treatment

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat a polyneuropathic pain. While the exampleillustrates the use of a PSCB to treat pain induced from chemotherapytreatment, any pain involving two or more peripherial nerves, such as,e.g., diabetic neuropathy, treatment-induced pain, postmastectomysyndrome. post-polio syndrome, diabetes, alcohol, amyloid, toxins, HIV,hypothyroidism, uremia, vitamin deficiencies, 2′,3′-didexoycytidine(ddC) treatment and Fabry's disease, can also be treated using thismethod.

A cancer patient undergoing chemotherapy presents symptoms that arediagnosed as chemotherapy-induced pain. That patient is treated orallywith a therapeutically-effective amount of a pharmaceutically acceptablecomposition comprising a PSCB. Within one day after the administrationof a PSCB therapy, the patient's pain is substantially alleviated.Repeated administration of the PSCB composition maintains this painrelief.

Example 12 Oral Administration of a Persistent Sodium Current Blocker toTreat Allodynia

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat allodynia.

A patient presents pain symptoms that are diagnosed as allodynia. Sheindicates that whenever something gently touches her lest forearm, shefeels an intense pain like a sudden burning sensation. That patient istreated orally with a therapeutically-effective amount of apharmaceutically acceptable composition comprising a PSCB. Within oneday after the administration of a PSCB therapy, the patient's pain issubstantially alleviated. Repeated administration of the PSCBcomposition maintains this pain relief.

Example 13 Oral Administration of a Persistent Sodium Current Blocker toTreat Hyperalgesia

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat hyperalgesia.

A patient presents pain symptoms that are diagnosed as hyperalgesia. Heindicates that whenever he mildly bumps his right thigh against a hardobject, like a table corner, a great shooting pain occurs to such anextent that he needs to sit down. That patient is treated orally with atherapeutically-effective amount of a pharmaceutically acceptablecomposition comprising a PSCB. Within one day after the administrationof a PSCB therapy, the patient's pain is substantially alleviated.Repeated administration of the PSCB composition maintains this painrelief.

Example 14 Oral Administration of a Persistent Sodium Current Blocker toTreat Hyperpathia

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat hyperpathia.

A patient presents pain symptoms that are diagnosed as hyperpathia. Thatpatient is treated orally with a therapeutically-effective amount of apharmaceutically acceptable composition comprising a PSCB. Within oneday after the administration of a PSCB therapy, the patient's pain issubstantially alleviated. Repeated administration of the PSCBcomposition maintains this pain relief.

Example 15 Oral Administration of a Persistent Sodium Current Blocker toTreat Chronic Pain from a Migraine Headache Pain

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat pain from a headache. While the exampleillustrates the use of a PSCB to treat pain resulting from a migraineHeadache, any headache pain, such as, e.g., tension-type headache,cluster headache, hormone headache, rebound headache, sinus headache andorganic headache, can also be treated using this method.

A patient presents pain symptoms that are diagnosed as resulting from amigraine headache. That patient is treated orally with atherapeutically-effective amount of a pharmaceutically acceptablecomposition comprising a PSCB. Within one day after the administrationof a PSCB therapy, the patient's pain is substantially alleviated.Repeated administration of the PSCB composition maintains this painrelief.

Example 16 Oral Administration of a Persistent Sodium Current Blocker toTreat Pain Associated with Rheumatoid Arthritis

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat chronic pain resulting from inflammatorydisorder. While the example illustrates the use of a PSCB to treatchronic pain resulting from a rheumatoid arthritis, any inflammatorydisorder-induced pain, such as, e.g., osteoarthritis, gouty arthritis,spondylitis or autoimmune diseases such as lupus erythematosus, can alsobe treated using this method.

A patient presents pain symptoms that are diagnosed as resulting fromrheumatoid arthritis. That patient is treated orally with atherapeutically-effective amount of a pharmaceutically acceptablecomposition comprising a PSCB. Within one day after the administrationof a PSCB therapy, the patient's pain is substantially alleviated.Repeated administration of the PSCB composition maintains this painrelief.

Example 17 Oral Administration of a Persistent Sodium Current Blocker toTreat Chronic Back Pain

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat chronic pain resulting from excessive muscletension. While the example illustrates the use of a PSCB to treatchronic lower back pain, any excessive muscle tension-induced pain canalso be treated using this method.

A patient presents with a non-spasmodic muscle pain localized at thelumbar region of the back due to a herniated disc. That patient istreated orally with a therapeutically-effective amount of apharmaceutically acceptable composition comprising a PSCB. Within oneday after the administration of a PSCB therapy, the patient's pain issubstantially alleviated. Repeated administration of the PSCBcomposition maintains this pain relief.

Example 18 Oral Administration of a Persistent Sodium Current Blocker toPain Associated with Treat Irritable Bowel Syndrome

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat pain resulting from chronic gastrointestinalinflammations. While the example illustrates the use of a PSCB to treatthe pain associated with irritable bowel syndrome, any gastrointestinalinflammation-induced pain, such as, e.g., Crohn's disease, ulcerativecolitis and gastritis, can also be treated using this method.

A patient presents pain symptoms that are diagnosed as resulting fromirritable bowel syndrome. That patient is treated orally with atherapeutically-effective amount of a pharmaceutically acceptablecomposition comprising a PSCB. Within one day after the administrationof a PSCB therapy, the patient's pain is substantially alleviated.Repeated administration of the PSCB composition maintains this painrelief.

Example 19 Oral Administration of a Persistent Sodium Current Blocker toTreat Post-Operative Pain

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat post-operative pain.

A patient presents pain symptoms resulting from a surgical operation.That patient is treated orally with a therapeutically-effective amountof a pharmaceutically acceptable composition comprising a PSCB. Withinone day after the administration of a PSCB therapy, the patient's painis substantially alleviated. Administration of the PSCB compositioncontinues for about 1 to about 4 weeks to maintain this pain relief.

Example 20 Oral Administration of a Persistent Sodium Current Blocker toTreat Pain Associated with Fibromyalgia

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat pain associated with fibromyalgia.

A patient presents pain symptoms that are diagnosed as resulting fromfibromyalgia. That patient is treated orally with atherapeutically-effective amount of a pharmaceutically acceptablecomposition comprising a PSCB. Within one day after the administrationof a PSCB therapy, the patient's pain is substantially alleviated.Repeated administration of the PSCB composition maintains this painrelief.

Example 21 Oral Administration of a Persistent Sodium Current Blocker toTreat Pain Associated with Repetitive Motion Disorder of the Wrist

This example shows a method of persistent sodium current blocker (PSCB)therapy using a pharmaceutically acceptable composition comprising aPSCB compound to treat pain resulting from repetitive motion disorders(RMDs). While the example illustrates the use of a PSCB to treat thepain associated with RMDs of the wrist, any RMD-induced pain occurringin, e.g., hands, elbows, shoulders, neck, back, hips, knees, feet, legs,and ankles, can also be treated using this method.

A patient presents pain symptoms that are diagnosed as resulting from anRMD of the wrist. That patient is treated orally with atherapeutically-effective amount of a pharmaceutically acceptablecomposition comprising a PSCB. Within one day after the administrationof a PSCB therapy, the patient's pain is substantially alleviated.Repeated administration of the PSCB composition maintains this painrelief.

Although the present invention has been described with reference to thedisclosed embodiments, one skilled in the art will readily appreciatethat the specific experiments disclosed are only illustrative of thepresent invention. Various modifications can be made without departingfrom the spirit of the present invention.

1. A method of treating an epileptic condition in a mammal, comprisingadministering to said mammal an effective amount of a selectivepersistent sodium channel antagonist, wherein said antagonist has atleast 20-fold selectivity for a persistent sodium current relative to atransient sodium current, and wherein said antagonist is a compoundincluded in formula 3, or a pharmaceutically acceptable salt, ester,amide, stereoisomer or racemic mixture thereof:

wherein, Ar⁷ is phenyl or a substituted phenyl; X is O; R¹⁷ and R¹⁸ areindependently selected from the group consisting of hydrogen, hydroxy,and a C₁ to C₈ alkyl; R¹⁹ and R²⁰ are independently selected from thegroup consisting of hydrogen, hydroxy, and a C₁ to C₈ alkyl; R²¹ isselected from the group consisting of hydrogen, hydroxy, and a C₁ to C₈alkyl; R is

a is 0 or 1; and m is 0, 1, 2, or
 3. 2. The method of claim 1, whereinsaid persistent sodium current is Na_(v)1.1 persistent current.
 3. Themethod of claim 1, wherein said persistent sodium current is Na_(v)1.2persistent current.
 4. The method of claim 1, wherein said persistentsodium current is Na_(v)1.3 persistent current.
 5. The method of claim1, wherein said persistent sodium current is Na_(v)1.5 persistentcurrent.
 6. The method of claim 1, wherein said persistent sodiumcurrent is Na_(v)1.6 persistent current.
 7. The method of claim 1,wherein said persistent sodium current is Na_(v)1.7 persistent current.8. The method of claim 1, wherein said persistent sodium current isNa_(v)1.8 persistent current.
 9. The method of claim 1, wherein saidpersistent sodium current is Na_(v)1.9 persistent current.
 10. Themethod of claim 1, wherein said mammal is a human.
 11. The method ofclaim 1, wherein said antagonist has at least 50-fold selectivity forsaid persistent sodium current relative to said transient sodiumcurrent.
 12. The method of claim 1, wherein said antagonist has at least200-fold selectivity for said persistent sodium current relative to saidtransient sodium current.
 13. The method of claim 1, wherein saidantagonist has at least 400-fold selectivity for said persistent sodiumcurrent relative to said transient sodium current.
 14. The method ofclaim 1, wherein said antagonist has at least 600-fold selectivity forsaid persistent sodium current relative to said transient sodiumcurrent.
 15. The method of claim 1, wherein said antagonist has at least1000-fold selectivity for said persistent sodium current relative tosaid transient sodium current.
 16. The method of claim 1, wherein saidantagonist is administered peripherally.
 17. The method of claim 1,wherein said antagonist is administered systemically.
 18. The method ofclaim 1, wherein said antagonist is administered orally.
 19. The methodof claim 1, wherein said antagonist is administered in a sustainedrelease formula.
 20. The method of claim 1, wherein said antagonist isadministered in an bioerodible delivery system.
 21. The method of claim1, wherein said antagonist is administered in a non-bioerodible deliverysystem.
 22. The method of claim 1, wherein said Ar⁷ is phenyl.
 23. Themethod of claim 1, wherein said Ar⁷ is a substituted phenyl.
 24. Themethod of claim 23, wherein said substituted phenyl is substituted withone or more of halogen, a C₁ to C₈ alkyl, NO₂, CF₃, OCF₃, OCF₂H, CN, or(CR⁵R⁶)_(c)N(R⁷)₂, wherein, R⁵ and R⁶ are independently selected fromthe group consisting of hydrogen, hydroxy, fluoro, and a C₁ to C₈ alkyl;R⁷ is selected from the group consisting of hydrogen, and a C₁ to C₈alkyl; and c is 0, 1, 2, 3, 4, or
 5. 25. The method of claim 1, whereinsaid R¹⁷ is hydrogen, methyl, ethyl, propyl, or isopropyl.
 26. Themethod of claim 1, wherein said R¹³ is hydrogen, methyl, ethyl, propyl,or isopropyl.
 27. The method of claim 1, wherein said R¹⁹ is hydrogen,methyl, ethyl, propyl, or isopropyl.
 28. The method of claim 1, whereinsaid R²⁰ is hydrogen, methyl, ethyl, propyl, or isopropyl.
 29. Themethod of claim 1, wherein said R²¹ is hydrogen, methyl, ethyl, propyl,or isopropyl.
 30. The method of claim 1, wherein said R²² is hydrogen,methyl, ethyl, propyl, or isopropyl.
 31. The method of claim 1, whereinsaid R²³ is hydrogen, methyl, ethyl, propyl, or isopropyl.
 32. Themethod of claim 23, wherein said antagonist is6-Isopropyl-3-methyl-2-{4-[(4-propoxy-benzylidene)-amino]-benzylidene}-cyclohexanone.33. The method of claim 23, wherein said antagonist is


34. The method of claim 1, wherein said neuropathic pain is a neuralgia.35. The method of claim 34, wherein said neuralgia is selected from thegroup consisting of a trigeminal neuralgia, a post-herpetic neuralgia, aglossopharyngeal neuralgia, a sciatica and an atypical facial pain. 36.The method of claim 1, wherein said neuropathic pain is adeafferentation pain syndrome.
 37. The method of claim 36, wherein saiddeafferentation pain syndrome is selected from the group consisting ofan injury to the brain or spinal cord, a post-stroke pain, a phantompain, a paraplegia, a peripheral nerve injury, a brachial plexusavulsion injury and a lumbar radiculopathy.
 38. The method of claim 1,wherein said neuropathic pain is a complex regional pain syndrome(CRPS).
 39. The method of claim 38, wherein said complex regional painsyndrome is selected from the group consisting of a reflex sympatheticdystrophy (CRPS Type I) and a causalgia (CRPS Type II).
 40. The methodof claim 1, wherein said neuropathic pain is a polyneuropathic pain. 41.The method of claim 40, wherein said complex regional pain syndrome isselected from the group consisting of a diabetic neuropathy, achemotherapy-induced pain, a treatment-induced pain, and apostmastectomy syndrome.
 42. The method of claim 1, wherein saidneuropathic pain is a centrally-generated neuropathic pain.
 43. Themethod of claim 42, wherein said centrally-generated neuropathic pain isselected from the group consisting of a dorsal root ganglioncompression, an inflammation of the spinal cord, a contusion, a tumor ofthe spinal cord, a hemisection of the spinal cord, a tumor of thebrainstem, a tumor of the thalamus, a tumor of the cortex, a trauma ofthe brainstem, a trauma of the thalamus and a trauma of the cortex. 44.The method of claim 1, wherein said neuropathic pain is aperipherially-generated neuropathic pain.
 45. The method of claim 44,wherein said peripherially-generated neuropathic pain is selected fromthe group consisting of a neuroma, a nerve compression, a nerve crush, anerve stretch, a nerve entrapment and an incomplete nerve transsection.46. The method of claim 1, wherein said neuropathic pain is anallodynia, a hyperalgesia amd a hyperpathia.
 47. The method of claim 1,wherein said effective amount reduces the symptoms of neuropathic painby at least 30%.
 48. The method of claim 1, wherein said effectiveamount reduces the symptoms of neuropathic pain by at least 50%.
 49. Themethod of claim 1, wherein said effective amount reduces the symptoms ofneuropathic pain by at least 70%.
 50. The method of claim 1, whereinsaid effective amount reduces the symptoms of neuropathic pain by atleast 90%.